Amphipatic polycarboxylic chelates and complexes with paramagnetic metals as MRI contrast agents

ABSTRACT

Substituted polycarboxylic ligand molecules and corresponding metal complexes of said ligands, preferably paramagnetic metals complexes for generating responses in the field of magnetic resonance imaging (MRI)  
     The paramagnetic complexes of the polycarboxylic ligands possess advantageous tensioactive properties and are useful as MRI contrast media in formulations for investigating the blood pool.

FIELD OF THE INVENTION

[0001] The present invention addresses novel polycarboxylic ligandmolecules and chelated complexes of said ligands with metals; the metalsare for instance transition metals, e.g. paramagnetic metals forgenerating responses in the field of magnetic resonance imaging (MRI)

[0002] The present polycarboxylic ligands exhibit outstandingtensioactive properties which make them particularly useful in the formof paramagnetic chelates for making formulations and compositions usefulas MRI contrast media of controllable and long lasting activity in theblood pool.

BACKGROUND ART

[0003] U.S. Pat. No. 5,466,438 (WO 92/231017) discloses compounds offormulae

[0004] in which formulae I-III:

[0005] R₁ is independently a substituted or unsubstituted C₇₋₃₀ straightchain or cyclic compound;

[0006] R₂ is independently a substituted or unsubstituted C₁-C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₃ or S, where R₃ is a C₁-C₃ alkyl;

[0007] n is 0-1 in formula I and 1-20 in formula III;

[0008] m is 1-2;

[0009] B is a substituted or unsubstituted C₁-C₃₀ straight chain orcyclic compound which may be internally interrupted by O, NH, NR₃ or S.

[0010] In formula IV:

[0011] R₁, R₂ are independently H or a substituted or unsubstitutedC₇-C₃₀ straight chain or cyclic compound;

[0012] R₃, R₄ are independently H or a substituted or unsubstitutedC₁-C₃₀ straight chain or cyclic compound which may be internallyinterrupted by O, NH, NR₅ or S, where R₅ is a C₁-C₃ alkyl;

[0013] and in formula V:

[0014] R₁ is independently a substituted or unsubstituted C₇-C₃₀straight chain or cyclic compound

[0015] R₂ is independently a substituted or unsubstituted C₁-C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₄ or S, where R₄ is a C₁-C₃ alkyl;

[0016] R₃ is independently a substituted or unsubstituted C₁-C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₄ or S, where R₄ is a C₁-C₃ alkyl and

[0017] m is 0-12.

[0018] The reference also discloses contrast agents obtained with thecompounds of the above formulae (I-V), the latter further comprisinglipids; the lipids are in the form of emulsions, liposomes or micelles.

[0019] U.S. Pat. No. 5,312,617 dicloses a method of imaging comprisingadministering to patients a contrast agent comprising a complex of aparamagnetic metal and a ligand selected from formulae IV and Vdisclosed in the foregoing U.S. Pat. No. 5,466,438.

[0020] Liposomes incorporating the above chelates are also disclosed aswell as the possibility of having the compounds in the form of emulsionsor micelles.

[0021] The micelles can be prepared by a variety of conventionalliposome preparatory techniques; suitable lipids include, for example,monomyristoyl-phosphatidyl-choline, monopalmitoyl-phosphatidylcholine,dibutyroyl-phosphatidylcholine and the like, linoleic acid, oleic acid,palmitic acid, and the like.

[0022] Lipid emulsions can be prepared by conventional techniques, forinstance a typical method is as follows:

[0023] 1. In a suitable flask, the lipids are dissolved in ethanol orchloroform or any other suitable organic solvent.

[0024] 2. The solvent is evaporated leaving a thin layer of lipid at thebottom of the flask.

[0025] 3. The lipids are resuspended in an aqueous medium, such asphosphate buffered saline, this producing an emulsion

[0026] 4. Sonication or microfluidization can then be applied to improvehomogeneity.

[0027] 5. The contrast agents can be added to the lipids duringpreparation of the emulsion, or they may be added to the emulsionafterwards.

[0028] 6. Useful additives include, for example, soybean lecithin,glucose, Pluronic F-68 and D,L-α-tocopherol; these additives areparticularly useful where injectable intravenous formulations aredesired.

[0029] The foregoing contrast agents may further comprise suspensionstabilizers such as polyethyleneglycol, lactose, mannitol, sorbitol,ethyl alcohol, glycerin, lecithin, polyoxyethylene sorbitan monoleate,sorbitan monoleate and albumin. Various sugars and other polymers mayalso be added, such as polyethylene glycol, polyvinylpyrrolidone,polypropylene glycol and polyoxyethylene.

[0030] The contrast agents of this reference have high T₁ and T₂relaxivity, especially when lipids are also present. Because of the highrelaxivity, these contrast media are particularly useful for imaging theblood pool.

SUMMARY OF THE INVENTION

[0031] Despite the merit of the paramagnetic polycarboxylic chelates ofthe prior art as contrast agents for MRI, there was a need for a newrange of chelating compounds of further improved properties designed toprovide blood-pool contrast agents of outstanding long life in thecirculation. In view of their structure including strongly hydrophobicand hydrophilic moieties, the compounds of the present invention achievea significant step in the right direction.

[0032] The novel compounds of the present invention, either racemic orenantiomeric, have the following formulae (\) and (IV)

[0033] in which n and m are 1 or 0 but not simultaneously 1, and

[0034] when n=m=0, R′ is H, and R* is a C₁₂₋₂₅ linear or ramified,saturated or unsaturated, hydrocarbon radical;

[0035] when n=1 and m=0, R* is H or a C₁₋₃ alkyl or alkylenesubstituent; and R′ is selected from —NHR₃, —NR₄R₅ and —OR₆ where the R₃to R₆ are independently C₁₋₂₅ linear or ramified, saturated orunsaturated, hydrocarbon radicals optionally interrupted by —CO—and/or—O—and optionally terminated by —NR₇R₈ in which R₇ and R₈ areindependently H or C₁₂₋₂₅ hydrocarbon radicals;

[0036] when n=0 and m=1, R* is H or a C₁₋₃ alkyl or alkylenesubstituent; and R′ is selected from R₉ and —CH₂—O—CO—R₉ in which R₉ isa C₁₀₋₃₀ linear or ramified, saturated or unsaturated, hydrocarbonradical optionally interrupted by —NH—, —NR₁₀—, —CO— or —O—, R₁₀ being alower aliphatic hydrocarbon; and

[0037] R₁₂ is H or a C₁₂₋₃₀ hydrocarbon radical optionally interruptedby —NH—, —NR₁₀—, —CO—or —O—and optionally terminated by a cholesterylresidue, and the R₁₃ are —OH; or one or two R₁₃ are a —NH—R₁₄ group inwhich R₁₄ is a C₂₋₃₀ linear or ramified, saturated or unsaturated,hydrocarbon radical optionally interrupted by —NH—, -NR₁₀—, —CO—, —O—,and/or —OPO(OH)O—, the remaining R₁₃ being —OH.

[0038] The compounds of formulae (\) and (IV) can be used as chelates ofparamagnetic metals, preferably Gd(III), Mn(II), Cr(III), Cu(II),Fe(III), Pr(III), Nd(III), Sm(III), Tb(III), Yb(III), Dy(III), Ho(III)and Er(III) in the preparation of MRI contrast formulations andcompositions of outstanding long life in the blood which makes themideal agents for investigating the circulation in appended organs.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Preferred are the compounds encompassed by formula (\), in whichn and m are 1 or 0 but not simultaneously 1, and when n=1 and m=0, R* isan alkylene group, and the other variable groups are the meaningsdefined above.

[0040] Equally preferred are the compounds, encompassed by formula (\),in which n and m are 1 or 0 but not simultaneously 1, and when n=1 andm=0, R* is H or a C₁₋₃ alkyl; and R′ is selected from —NHR_(3,) or—NR₄R₅, where the R₃ to R₅ groups are independently C₁₂₋₂₅ linear orramified, saturated or unsaturated, hydrocarbon radicals, optionallyinterrupted by —CO—and/or —O—and optionally terminated by —NR₇R₈ inwhich R₇ and R₈ are independently H or C₁₂₋₂₅ hydrocarbon radicals withthe same meaning just above defined.

[0041] Equally preferred are the compounds encompassed by formula (\),in which n and m are 1 or 0 but not simultaneously 1, and when n=0 andm=1, R* is an alkylene group, and the other variable groups are themeaning defined above.

[0042] Equally preferred are the compounds encompassed by formula (\),in which n and m are 1 or 0 but not simultaneously 1, and when n=1 andm=0, R* is H or a C₁₋₃ alkyl;and R′ is selected from R₉ and —CH₂—O—CO—R₉in which R₉ is a C₁₂₋₂₅ linear or ramified, saturated or unsaturated,hydrocarbon radical optionally interrupted by —NH—, —NR₁₀—, —CO—or —O—,R₁₀ being a C₁₋₄ linear or ramified, saturated or unsaturated,hydrocarbon radical.

[0043] Furthermore are prefered the compounds of general formula (IV) inwhich R₁₂ is H, and two R₁₃ are a —NH—R₁₄ group in which R₁ ₄ is aC₁₂₋₂₅ linear or ramified, saturated or unsaturated, hydrocarbon radicaloptionally interrupted by —NH—, —NR₁₀—, —CO—, —O—, and/or —OPO(OH)O—,the remaining R₁₃ being —OH.

[0044] The compounds of formula (\) are preferably selected amongcompounds of the following formulae (I), (II) or (III)

[0045] wherein R* is as defined heretofore;

[0046] R is H or a C₁₋₃ alkyl or alkylene substituent;

[0047] R₁ is selected from —NHR₃, —NR₄R₅ and —OR₆ where the R₃ to R₆ areindependently C₁₋₂₅ linear or ramified, saturated or unsaturated,hydrocarbon radicals optionally interrupted by —CO—and/or —O—andoptionally terminated by —NR₇R₈ in which R₇ and R₈ are independently Hor C₁₂₋₂₅ hydrocarbon radicals;

[0048] R₂ is selected from R₉ and —CH₂—O—CO—R₉ in which R₉ is a C₁₀₋₆₀linear or ramified, saturated or unsaturated, hydrocarbon radicaloptionally interrupted by one or more —NH—, —NR₁₀—, —CO—or —O—, R₁₀being a lower aliphatic hydrocarbon.

[0049] For instance, the compounds of formula (II) can have formula(IIa)

[0050] in which R₃ is a C₁₂₋₂₅ linear or ramified, saturated orunsaturated, hydrocarbon radical.

[0051] Particularly preferred are the compounds of formula (IIa) inwhich R₃ is a C₁₆₋₂₀ linear or ramified, saturated or unsaturated,hydrocarbon radical.

[0052] Or they can have formula (IIb)

[0053] in which R₄ and R₅ are independently C₁₂₋₂₅ linear or ramified,saturated or unsaturated, hydrocarbon radicals optionally interrupted by—CO—and/or —O—.

[0054] Particularly preferred are the compounds of formula (IIb) inwhich R₄ and R₅ are independently C₁₆₋₂₀ linear or ramified, saturatedor unsaturated, hydrocarbon radicals interrupted by —CO—and —O—.

[0055] Furthermore, they can have formula (IIc)

[0056] in which A is —NH—or —O—, A₁ is a C₁₋₂₀ linear or ramified,saturated or unsaturated, hydrocarbon radicals optionally interrupted by—CO—and/or —O—and R₇ and R₈ are defined as above.

[0057] Particularly preferred are the compounds of formula (IIc) inwhich R is an alkylene substituent.

[0058] Equally preferred are the compounds of formula (IIc) in which Ais —NH—, A₁ is a C₁₋₂₀ linear or ramified, saturated or unsaturated,hydrocarbon radicals interrupted by —CO—and —O—and R₇ and R₈ are definedas above. Also the compounds of formula (IIc) are preferred in which Ais —O—and A₁ is a C₁₋₂₀ linear or ramified, saturated or unsaturated,hydrocarbon radicals interrupted by —CO—and —O—and R₇ and R₈ are definedas above.

[0059] Compounds of formula (III) can have formula (IIIa)

[0060] in which R is H and R₂ is is a C₁₀₋₃₀ linear or ramified,saturated or unsaturated, hydrocarbon radical optionally interrupted byone or more —NH—, —N—, —CO—or —O. Otherwise, they can have formula(IIIb) below

[0061] in which R is H and R₉ is a C₁₀₋₂₅ linear alkyl, or a C₁₀₋₅₀linear or ramified, saturated or unsaturated, hydrocarbon radicaloptionally interrupted by one or more —N—, —CO—and/or —O—.

[0062] Some preferred compounds of formula (IV) are those in which allthe R₁₃ are —OH and R₁₂ is defined as mentioned above. Otherwise,compounds of formula (IV) can be selected from the compounds of formulae(IVa) and (IVb) below. In Formula (IVa),

[0063] the R₁₄ are independently as defined above for formula (IV).

[0064] Particularly preferred are the compounds of formula (Iva) inwhich R₁₄ is C₁₂₋₂₅ linear or ramified, saturated or unsaturated,hydrocarbon radical optionally interrupted by —CO—and/or—O—, and/or-OPO(OH)O—.

[0065] In formula (IVb) shown below,

[0066] R₁₄ is a C₁₂₋₂₅ linear or ramified, saturated or unsaturated,hydrocarbon radical.

[0067] The compounds of this invention of formulae (\) and (IV) can beas represented in the formulae, or they can be in the form of complexchelates with paramagnetic metal ions (as indicated heretofore) and thesalts thereof with physiologically acceptable bases selected fromprimary, secondary, tertiary amines and basic aminoacids, or inorganichydroxides of sodium, potassium, magnesium, calcium or mixtures thereof;

[0068] or with physiologically acceptable anions of organic acidsselected from acetate, succinate, citrate, fumarate, maleate, oxalate,or inorganic acids selected from hydrogen halides, sulphates,phosphates, phosphonates and the like;

[0069] or with cations or anions of aminoacids selected from lysine,arginine, ornithine, aspartic and glutamic acids, and the like;

[0070] For preparing the compounds of formula (I) in the form ofcomplexes with metals (ME), one can proceed as in the following Scheme 1

[0071] Where Pg is a protecting group;

[0072] R* is as defined for formula (I);

[0073] ME^(n+) is a metal ion;

[0074] n=2 or 3.

[0075] In step al the compound 1A, i.e.1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, whose carboxylicgroups are suitably protected by groups such as benzyl or t-butyl, isreacted with R*—X, where R* is a substituent residue and X is a leavinggroup such as Cl, Br, I. The reaction product is then deprotected byknown methods (e.g. with CF₃COOH) to give the free ligand 1B. The ligandis then complexed with a suitable metal ion oxide or salt (preferablyparamagnetic), such as Gd oxide, chloride or acetate, in order to obtainthe desired metal complex chelate 1C. Depending on the value of n, 1Cmay be salified with a suitable counter-ion.

[0076] For the complexes of the compounds of formula (IIa), one mayproceed according to the scheme 2 below:

[0077] in which Pg is a protecting group as in Scheme 1;

[0078] R and R₃ have been defined above; and ME and n are as in Scheme1.

[0079] According to the method of Scheme 2, one prepares compound 2C bythe reaction of a halogenated halide (or equivalent) with a primaryamine R₃NH₂ in a suitable solvent, such as CH₂Cl₂, CHCl₃ or H₂O/CH₂Cl₂mixtures, in the presnce of a base (e.g. K₂CO₃). Then, Compound 2C isreacted with compound 2D and the product is deprotected (b2) to furnishthe desired free ligand 2E. The latter is finally complexed according tothe general procedure disclosed in Scheme 1. If required, i.e. dependingon whether n has an appropriate value, compound 2F may be salified witha suitable counter-ion.

[0080] For preparing the metal complexes of the compounds of formula(IIb), one may proceed according to the Scheme 3 below:

[0081] in which n=1-6; p=0-5; m=2 or 3; Y=halogen; Pg is a protectinggroup and Alk is a lipophilic alkyl chain.

[0082] In step a3 compound 3A is reacted with a suitable long-chaincarboxylic acid halide 3B in a suitable aprotic dipolar solvent toobtain compound 3C. The latter is reacted (step b3) with compound 3D,i.e. 1,4,7,10-tetraazα-cyclododecane-1,4,7,10-tetraacetic acid of whichthree acetic groups are protected with, for example, benzyl or t-butylgroups, in the presence of 1-propanephosphonic acid cyclic anhydride(PPAA) and a base (e.g. Et₃N) in a suitable solvent such as CH₂Cl₂.

[0083] Compound 3E obtained in step C₃ is deprotected by known methods(e.g. by catalytic hydrogenation) which provides the free ligand 3F,which is then complexed with a metal (step d3), according to theprocedure described earlier. This affords the desired complex chelate3G. Then, compound 3G may be salified with a suitable counter-ion if thevalue of m permits.

[0084] For preparing the compounds of formula (Iic) in which A is —NH—,one can proceed like in the previous scheme, the first step(condensation of triprotected1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (4A) with anamine H₂N—A₁—NR₇R₈ (4B) being effected in the presence, as condensing aagent, of (benzotriazo-1-yloxy)-tris(dimethylamino)-phosphoniumhexafluorophosphate and a sterically hindered tertiary amine such asdiisopropylethylamine (DIEA) when 4B is an α-aminoacid derivative, orN,N′-bis(2-oxo-3-oxazolidyl)-phosphoro-diamidic chloride (BOP). This isillustrated in Scheme 4.

[0085] A similar method can be applied for compounds (IIc) in which A is—O—, i.e. the esterification of triprotected1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with ahalogenated compound X—A₁—NR₇R₈ in the presence of1,8-diazabicyclo[5.4.0]undecene. Then the resulting intermediate 5C isdeprotected, complexed and salified as in the previous schemes (Scheme5).

[0086] The compounds of formula (IIIa) can be obtained as illustrated inScheme 6. The protected 1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid 6A is reacted with an alkyl epoxide

[0087] in a solvent such as ethanol and the resulting product isdeprotected and treated as described in the previous schemes

[0088] A possible technique for making the compounds (IIIb) and thecorresponding metal chelates and salts is illustrated in Scheme 7. Herethe initial reactant to be condensed with the triprotected1,4,7,10-tetraaza-cyclododecane-1,4,7-triaacetic acid is2,3-epoxypropanol 7B which provides the vic-diol 7C, the primary —OH ofwhich is thereafter esterified with an acid R₉—COOH, the remaining stepsbeing as described previously.

[0089] The following preparative methods are applicable regarding thecompounds of formula (IV) in which R₁₂ is H. For instance, for thecompounds (IVa), one may operate as illustrated in Scheme 8, by reactingin DMF the DTPA cyclic dianhydride(N,N-bis[2-(2,6-dioxo-4-morpholinyl)ethyl]gly-cine) 8A with an amineH₂NR₁₄, the remaining step being that of complexation with a metal andpossible salification as discussed earlier.

[0090] Otherwise, one may first effect protection of up to four of the—COOH's groups in DTPA, the unprotected group being thereafter amidatedin DMF with an amine H₂NR₁₄ according to usual means (see Scheme 9).

[0091] One preparative route for the compounds of formula (IV) with asubstituent R₁₂ in α- to the central carboxylic function is to firstattach in the said position, a carbon chain functionalized with, forexample, a —NH₂ or —COOH group (Scheme 10). For example, compound 10Acan be prepared, according to Rapoport et al. in J. Org. Chem. 58(1993), 1151-1158, or using a method disclosed in WO 98/05626. Thesynthon is then reacted with, for example, a chloride of a carboxylicacid having the desired chain length, or with a suitable amine,depending on the nature of the said functional group. Then, theresulting compound 10B is deprotected and complexed as already shown inthe previous Schemes.

[0092] This technique is exemplified in the synthesis of the compound ofExample 16 involving reaction with cholesteryl chloroformate.

[0093] The injectable compositions and formulations according to theinvention which are usable as contrast agents for MRI investigationswill preferably contain further additives, in addition to one or more ofthe afore discussed novel paramagnetic chelates and a carrier liquid.The additives include non-ionic and/or ionic surfactants and mixturesthereof, as well as other amphipatic compounds. Due to theirphysiological suitability, the non-ionic surfactants are preferred. Thenon-ionic surfactants are preferably block-copolymers havingpolyoxyethylene and polyoxypropylene sequences,polyethyleneglycol-alkylethers such as, for example,polyethyleneglycol-octadecylether, or polyoxyethylene fatty acid estersor polyoxyethylene sorbitan fatty acid esters, or n-alkylglycopyranoside and n-alkyl maltotrioside. The non-ionic surfactant inthe compositions of the invention is conveniently selected from thecommercially available products, such as Pluronic®, Poloxamer®,Poloxamine®, Synperonic®, BRIJ®, Myrj®, Tween®s (polysorbates) and theirmixtures. The weight proportion of the surfactant relative to the amountof the paramagnetic imaging agent is from 1:50 to 50:1, preferably 1:10to 10:1, and even more preferably 1:1. The ionic surfactants preferablyinclude biliary acid salts such as sodium deoxycholate.

[0094] The amphipatic compounds suitable in the present compositions arephospholipids which may be selected from phosphatidic acid (PA),phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol(PI), cardiolipin (CL) and sphyngomielin (SM). The amphipatic compoundmay also consists of a monophosphate ester of a substituted or partiallysubstituted glycerol, at least one functional group of said glycerolbeing esterified by saturated or unsaturated aliphatic fatty acid, oretherified by saturated or unsaturated alcohol, the other two acidicfunctions of the phosphoric acid being either free or salified withalkali or earth-alkali metals. Preferably the phosphate esters willinclude monophosphates of fatty acid glycerides selected fromdimiristoylphosphatidic acid, dipalmitoylphosphatidic acid, ordistearoylphosphatidic acid.

[0095] The phospholipids may also include diacyl and dialkylglycerophospholipids in which the aliphatic chains have at least twelvecarbon atoms, as well as one or more compounds selected from ionic andneutral phospholipids, monoalkyl or alkenyl esters of phosphoric acidand/or cholesterol, ergosterol, phytosterol, sitosterol, lanosterol,tocopherol. In the compositions containing phospholipids, the weightproportion of the phospholipids to the amphiphilic chelate seems notcritical and it may vary, for example, from 1:50 to 50:1. The practicalrange will be between 10:1 and 1:10, preferably between 1:5 and 5:1 andeven more preferably between 1:3 and 3:1. In the compositions in whichphospholipids are used the weight ratio of the phospholipid to thesurfactant may vary as above, however the ranges from 1:10 to 10:1 and,preferably, between 1:2 and 2:1 are considered optimal.

[0096] The compositions of the present invention may exist in micellarform, in which case they can be prepared using known techniques, namelyas described in WO 97/00087; Polym. Prepr. 1997, 38(1), 545-546; Acad.Radiol. 1996, 3, 232-238. These documents describe micelles ofamphiphilic Gd chelates useful in percutaneous lymphography. Themicelles have particle size between 10 and 500 nm, preferably between 50and 200 nm.

[0097] The micelles can be prepared in any physiologically acceptableaqueous liquid carrier, such as water or saline, neat or bufferd,according to usual practice. Depending upon the choice of thecomponents, the dispersion can be achieved by gentle mixing or by moreenergetic means, such as homogenisation, microfluidization orsonication.

[0098] In an advantageous mode of preparing the micelles of theinvention, one part by weight of the paramagnetic chelate contrastcomponent is admixed with one to two parts each of surfactants and oflipids, and with 100 to 200 parts of liquid carrier, for exampleTris/Glycerol buffer.

[0099] The compositions can be stored and used as such, or may belyophilized dry, according to known methods, e.g. by freeze-drying. Thisdry form (porous lumps or free flowing powder) is particularlyconvenient for long-term storage. The formulations can be reconstitutedbefore usage by dispersion of the lyophilizate in a physiologicallyacceptable liquid carrier, thus obtaining a suspension corresponding tothe early formulation and directly usable as NMR imaging contrast agent.

[0100] For practically applying the compositions of the invention in themedical field, the lyophilized components and the carrier liquid can bemarketed separately in a kit form. The lyophilized components may bestored under a dry, inert atmosphere ant the carrier liquid may furthercontain isotonic additives and other physiologically acceptableingredients, such as various mineral salts, vitamins, etc.

[0101] The compositions of the invention are particularly useful asmagnetic resonance contrast agents for the imaging of the blood pool.They have shown to possess a sufficiently high relaxivity effect on theblood after injection in the rat and an exceptionally favourableelimination kinetic profile from the blood circulation, as demonstratedby pharmacokinetic and biodistribution data. These two combinedcharacteristics make them very suitable for angiographic magneticresonance imaging in general. The compositions of the invention cantherefore facilitate MR angiography and help to assess myocardial andcerebral ischemia, pulmonary embolism, vascularization of tumours andtumour perfusion.

[0102] The following examples further illustrate the invention in moredetail.

EXAMPLE 1[10-Hexadecyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0103]

[0104] A) 10-Hexadecyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

[0105] A mixture of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidtris(1,1-dimethylethyl) ester (prepared according to EP-A-299795) (20.6g; 40 mmol) and 1-bromohexadecane (12.4 g; 40.6 mmol) in CH₃CN (500 mL)was heated to reflux for 2 h. Then, the reaction mixture was evaporatedand the residue was flash chromatographed (CH₂Cl₂/MeOH=9/1 (v/v) to givea solid. This product was dissolved in CHCl₃ and an excess CF₃COOH wasadded. After 2 h the reaction mixture was evaporated and the oilyresidue redissolved in CF₃COOH. After 16 h at room temperature thesolution was evaporated and the residue was purified by flashchromatography (CH₂Cl₂/MeOH/NH₄OH 25% (w/w)=12/4/1 (v/v/v)). The productwas dissolved in H₂O and acidified with 6N HCl; the solution was loadedonto an Amberlite^(□) XAD-8 resin column and eluted with a CH₃CN/H₂Ogradient.

[0106] The fractions containing the product were evaporated and driedunder reduced pressure to give the desired product (8.1 g; 14 mmol).Yield 35%. HPLC: 98% (area %). Karl Fisher (K. F.): 4.05%. The ¹³C—NMR,MS and IR spectra were consistent with the structure. Elemental analysis(%): C H N Calcd. 63.13 10.24 9.82 Found 63.16 10.53 9.84 anhydrous

[0107] B) [10-Hexadecyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0108] A solution of GdCl₃.6H₂O (3.9 g; 10.5 mmol) in H₂O (40 mL) wasadded to a solution of the product from the previous preparation (6 g;10.5 mmol) in H₂O; the pH was maintained at 6.8 by addition of asolution of NaOH 1N (30 mL) . The solution was treated with n-BuOH andthe organic phase was evaporated to give a solid that was extracted atreflux with CHCl₃ in a Soxhlet apparatus. The solution was evaporated togive a solid. The product was dissolved in H₂O and i-PrOH, loaded onto amixed bed of Amberlite^(□) IRA 400 (250 mL) and Duolite^(□) C20 MB resin(250 mL); then, it was and eluted with H₂O/i-PrOH 1:1. The fractionscontaining the product were evaporated to give the title compound (2 g;2.7 mmol).Yield 26%.

[0109] HPLC: 99% (area %); K.F.:2.49%; Weight loss: (120° C.): 10.45%.The MS and IR spectra were consistent with the structure postulated.Elemental analysis (%): C H N Gd Calcd. 49.70 7.65 7.73 21.69 Found49.80 7.95 7.69 21.49

EXAMPLE 2[10—Octadecyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]godolinium

[0110]

[0111] A) 10-Octadecyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

[0112] A mixture of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidtris(1,1-dimethylethyl)ester (37.5 g; 72.8 mmol) and 1-bromooctadecane(24.5 g; 73.5 mmol) in CH₃CN (500 mL) was heated to reflux. After 2 hthe reaction mixture was evaporated and the residue was dissolved inCHCl3 and a portion of CF₃COOH was added. After 16 h at room temperaturethe reaction mixture was evaporated and the oily residue dissolved inCF₃COOH. After 3 days at room temperature, the solution was evaporated,the residue taken up in CHCl₃ and the solution evaporated. Thisoperation was repeated three times. The oily residue was purified byflash chromatography as follows:

[0113] Eluents:

[0114] (a) CH₂Cl₂/MeOH=3/1 (v/v) 3 liters

[0115] (b) CH₂Cl₂/MeOH/NH₄OH 25% (w/w)=12/4/1 (v/v/v) 12 liters

[0116] (c) CH₂Cl₂/MeOH/NH₄OH 25% (w/w)=6/3/1 (v/v/v) 2 liters

[0117] The product was dissolved in H₂O and acidified with 6N HCl; then,the solution was loaded onto an Amberlite^(□) XAD-8 resin column andeluted with a CH₃CN/H₂O gradient. The product started eluting with 20%CH₃CN.

[0118] The fractions containing the product were evaporated and driedunder reduced pressure to give the desired product (24.2 g; 40.4 mmol).Yield 55%.

[0119] HPLC: 91% (area %); K.F.: 8.01%; the ¹³C—NMR, MS and IR spectrawere consistent with the structure. Elemental analysis (%): C H N Calcd.64.18 10.44 9.36 Found 64.17 10.48 9.33 anhydrous

[0120] B)[10-Octadecyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0121] (CH₃COO)₃Gd (7.05 g; 17.3 mmol) was added to a suspension of thefree ligand issued from the previous preparation (10.4 g; 17.3 mmol) inMeOH (400 mL) at 50° C. The reaction mixture was kept at 50° C. for 1 h,after which the clear solution was evaporated and dried under reducedpressure to give the title compound (11 g; 14.6 mmol). Yield 84%. HPLC:100% (area %); K.F.: 1.83%; Weight loss (120° C.) : 5.04%. The MS and IRspectra were consistent with the structure. Elemental analysis (%): C HN Gd Calcd. 51.04 7.90 7.44 20.88 Found 50.84 7.96 7.19 20.39

EXAMPLE 3[10-(2-Hydroxyoctadecyl)-1,4,7,10-tetraazacyclo-dodecane-1,4,7-triacetato-(3-)]gadolinium

[0122]

[0123] A)10-(2-Hydroxyoctadecyl)-1,4,7,10-tetraazacyclodode-cane-1,4,7-triaceticacid

[0124] A solution of 1,2-epoxyoctadecane (17.4 g; 65 mmol) in abs. EtOH(100 mL) was added dropwise to a solution of1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidtris-(1,1-dimethylethyl)ester (33.3 g; 65 mmol) in abs. EtOH and themixture was heated to reflux. After 3 h, the reaction mixture wasevaporated and the residue was dissolved in EtOAc and washed with brine.The organic phase was separated, dried over Na₂SO₄ and evaporated underreduced pressure. The residue was flash-chromatographed, (Eluent:CH₂Cl₂/MeOH=9/1 (v/v))

[0125] The obtained product was dissolved in 5N HCl (500 mL) and thesolution was heated to reflux. After 1.5 h, the mixture was evaporatedand the residue purified by flash chromatography CH₂Cl₂/MeOH/NH₄OH 25%(w/w)=12/4/1 (v/v/v))

[0126] The product was dissolved in H₂O and 6N HCl, the solution wasloaded onto an Amberlite® XAD-8 resin column (800 mL) and eluted with aCH₃CN/H₂O gradient.

[0127] The fractions containing the product were evaporated and driedunder reduced pressure to give the desired product (18 g; 29 mmol) Yield45%. Acidic titer (0.1 N NaOH): 95% HPLC: 94% (area %); K.F.: 7.10%. The¹³C-NMR, MS and IR spectra were consistent with the structure. Elementalanalysis (%): C H N Calcd. 62.51 10.16 9.11 Found 62.93 10.26 9.14anhydrous

[0128] B)[10-(2-Hydroxyoctadecyl)-1,4,7,10-tetraazacyclodode-cane-1,4,7-triacetato-(3-)]qadolinium

[0129] Gd₂O₃ (3.62 g.; 10 mmol) was added to a solution of the freeligand issued from the previous preparation (12.3 g; 20 mmol) in H₂O(150 mL) and the resulting suspension was heated at 50° C. for 40 h. Thereaction mixture was filtered through a Millipore^(□) apparatus (HA 0.45μm filter); the filtrate (pH 6.7) was evaporated under reduced pressureand dried to give the title compound (14.2 g.; 18 mmol). Yield 92%. Freeligand: 0.7%; HPLC 95% (area %); K.F.: 7.94%. The MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Gd Calcd.49.97 7.73 7.28 20.45 Found 49.98 7.88 7.29 20.57 Anhydrous

EXAMPLE 4[10-[2-(Octadecylamino)-2-oxoethyl]-1,4,7,10-tetraazacyclo)dodecane-1,4,7-triacetato(3-)]gadolinium

[0130]

[0131] A) 2-Bromo-N-octadecylacetamide (C.A.S. registry number15491-43-7)

[0132] A solution of bromoacetyl bromide (44.4 g; 0.22 mol) in CH₂Cl₂(50 mL) was added dropwise in 2.5 h at 20° C. to a mixture ofoctadecylamine (59.3 g; 0.22 mol) and K₂CO₃ (30.4 g; 0.22 mol) in CH₂Cl₂(600 mL) and H₂O (600 mL). After 16 h at room temperature the organiclayer was separated, washed with H₂O, dried over Na₂SO₄ and evaporated.The crude product was purified by flash chromatography(CH₂Cl₂/MeOH=100/1 (v/v)) to give the desired product (60 g; 0.154 mol).Yield 70%. GC: 96% (area %), K.F.: <0.1%; ¹H-NMR, ¹³C-NMR and MS spectrawere consistent with the postulated structure. Elemental analysis (%): CH N Br O Calcd. 61.52 10.33 3.59 20.46 4.09 Found 61.75 10.71 3.58 20.144.01

[0133] B)10-[2-(Octadecylamino)-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

[0134] A mixture of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidtris(1,1-dimethylethyl) ester (24 g; 46.6 mmol) and2-bromo-N-octadecylacetamide (18.2 g; 46.6 mmol) in EtOH (500 mL) washeated to reflux. After 2.5 h, the reaction mixture was evaporated, theresidue was dissolved in CH₂Cl₂ and CF₃COOH was added. After 15 min, thesolvent was evaporated and the oily residue dissolved in CF₃COOH. After16 h at room temperature the solution was evaporated and the oilyresidue was purified by flash chromatography (CH₂Cl₂/MeOH=3/1 (v/v);then CH₂Cl₂/MeOH/NH₄OH 25% (w/w)=12/4/1 (v/v/v)).

[0135] The product was dissolved in H₂O and 6N HCl, the solution wasloaded onto an Amberlite® XAD-8 resin column and eluted with a CH₃CN/H₂Ogradient. The product elutes with 50% CH₃CN.

[0136] The fractions containing the product were evaporated and driedunder reduced pressure to give the desired product (12 g; 18 mmol) Yield39%. Acidic titer (0.1 N NaOH): 91%; HPLC: 95% (area %); K.F.: 8.82%.The ¹³C-NMR, MS and IR spectra were consistent with the structure.Elemental analysis (%): C H N Calcd. 62.26 9.99 10.68 Found 62.28 9.6310.64 anhydrous

[0137] C)[10-[2-(Octadecylamino)-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0138] Gd₂O₃ (1.97 g; 5.4 mmol) was added to a solution of the freeligand from the previous preparation (7.12 g; 9.7 mmol) in H₂O (310 mL)and the resulting suspension was heated to 50° C. for 9.5 h. Thereaction mixture was filtered through a Millipore^(□) membrane (HA 0.45μm filter) and the solution was evaporated to give the title compound(8.6 g; 9.5 mmol). Yield 98%. HPLC: 98% (area %); K.F.: 9.98%; MS and IRspectra were consistent with the structure. Elemental analysis (%): C HN Gd Calcd. 50.41 7.71 8.64 19.41 Found 50.52 7.78 8.65 19.32 anhydrous

EXAMPLE 5[10-[2-(Dioctadecylamino)-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0139]

[0140] A) 2-Bromo-N,N-dioctadecylacetamide

[0141] This novel compound was prepared as follows: Bromoacetyl bromide(4.25 g; 21 mmol) was added dropwise to a solution of dioctadecylamine(10 g; 19 mmol) and Et₃N (2.13 g; 21 mmol) in CHCl₃ (400 mL). After 4 hat room temperature the reaction solution was washed with H₂O, driedover Na₂SO₄ and evaporated. The residue was purified by flashchromatography (n-hexane/Et₂O=8/2 (v/v) to give the desired product (7.5g; 11.5 mmol). Yield 61%. K.F.: <0.1%; The ¹H-NMR, ¹³C-NMR, MS and IRspectra were consistent with the postulated structure Elemental analysis(%): C H N Br Calcd. 70.99 11.91 2.18 12.43 Found 71.05 12.18 2.11 12.27

[0142] B)10-[2-(Dioctadecylamino)-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid trihydrochloride

[0143] A mixture of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidtris(1,1-dimethylethyl)ester (14.4 g; 28 mmol) and2-bromo—N,N-dioctadecylamide (18.1 g; 28 mmol) in EtOH (800 mL) washeated to reflux. After 3 h the reaction mixture was evaporated and theresidue was dissolved in CH₂Cl₂. The solution was washed with brine,dried over Na₂SO₄ and evaporated to give the crude alkylated ester. Thisproduct was suspended in 5N HCl and refluxed. After 2 h the suspensionwas filtered, the solid was washed with 5N HCl and dried under reducedpressure to give the desired compound (21.5 g; 21 mmol). Yield 75%.HPLC: 95.7% (area %). Argentometric titer (0.1 N AgNO₃): 98.5%; K.F.:4.79%. The ¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with thedesired structure. Elemental analysis (%): C H Cl N Calcd. 61.37 10.2910.45 6.88 Found 61.30 10.08 10.71 6.44 Anhydrous

[0144] C)[10-[2-(Dioctadecylamino)-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0145] A solution of GdCl₃.6H₂O (5.8 g; 15.7 mmol) in H₂O (50 mL) wasadded dropwise to a refluxing solution of the product from the previouspreparation (16 g; 15.7 mmol) and 1 N NaOH (94.3 mL; 94.3 mmol) in abs.EtOH (1 L) . After 1.5 h the mixture was cooled to room temperature,filtered and concentrated to half its volume, thus causing theprecipitation of a solid which was filtered, washed with H₂O and driedunder reduced pressure. The solid was purified by flash chromatography(CH₂Cl₂/MeOH/Et₃N=16/4/1 (v/v/v)) and then suspended in H₂O at 50° C.for 3 h. The suspension was drained and the solid was washed with H₂Oand dried under reduced pressure to give the title compound (11.5 g;10.8 mmol). Yield 69%. HPLC: 96% (area %); K.F.: 3.93%. The MS and IRspectra were consistent with the structure. Elemental analysis (%): C HN Gd Calcd. 58.78 9.30 6.59 14.80 Found 58.59 9.14 6.36 14.26 anhydrous

EXAMPLE 6[10-[2-Hydroxy-3-[(1-oxooctadecyl)oxy]propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0146]

[0147] A)10-(2,3-Dihydroxypropyl)-1,4,7,10-tetraazacyclodo-decane-1,4,7-triaceticacid tris(1,1-dimethylethyl)ester adduct with NaCl

[0148] A solution of 2,3-epoxypropanol (3.7 g; 50 mmol) in abs. EtOH (80mL) was added dropwise in 30 min to a refluxing solution of1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acidtris(1,1-dimethylethyl)ester (25.7 g; 50 mmol) in abs. EtOH (250 mL).After 2.5 h the solution was evaporated, the residue taken up with EtOAcand washed with brine. The organic phase was separated, dried (Na₂SO₄)and evaporated. The crude was purified by flash chromatography(CH₂Cl₂/MeOH—9/1 (v/v)) to give the desired compound (24 g; 37 mmol).Yield 74%. K.F.: 1.30%; The ¹³C-NMR, MS and IR spectra were consistentwith the desired structure. Elemental analysis (%): C H N Cl Na Calcd.53.82 8.72 8.66 5.48 3.55 Found 53.95 8.97 8.72 5.47 3.48 anhydrous

[0149] B)10-[2-Hydroxy-3-[(1-oxooctadecyl)oxy]propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid tris-(1,1-dimethylethyl)ester adduct with NaCl

[0150] A solution of dicyclohexylcarbodiimide (0.43 g; 2.1 mmol) inCHCl₃ (10 mL) was added dropwise in 10 min to a solution of stearic acid(0.44 g; 1.5 mmol) (commercial product),10-(2,3-dihydroxypropyl)-1,4,7,10-tetraazacyclo-dodecane-1,4,7-triaceticacid tris(1,1-dimethylethyl)ester adduct with NaCl (1 g; 1.5 mmol) and4-(dimethylamino)pyridine (0.06 g; 0.5 mmol) (commercial product) inCHCl₃ (40 mL) at 0° C. The temperature of the reaction mixture wasallowed to come back to normal. After 16 h, the mixture was concentratedto half its volume, the precipitate was filtered off and the solutionevaporated. The crude was purified by flash chromatography (CH₂Cl₂/MeOH=10/1 (v/v)) to give the desired compound (0.95 g; 1.04 mmol). Yield67%. The ¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with thestructure.

[0151] C)[10-[2-Hydroxy-3-[(1-oxooctadecyl)oxy]propyl]-1,4,7,10-tetraazacyclo-dodecane-1,4,7-triacetato(3-)]qadolinium

[0152] CF₃COOH (20 mL) was added to a solution of the product from theprevious preparation (13.9 g; 15.2 mmol) in CH₂Cl₂ (5 mL). After 24 hthe solution was evaporated and the residue takes up with more CF₃COOH(15 mL). After 6 h the mixture was evaporated and the crude desalted bydialysis (Spectra/Por® CE(Cellulose Ester) membrane MWCO 500) to afforda white solid (4 g). A portion of this solid (2.9 g) was dissolved in2-propanol (100 mL) and H₂O (25 mL) at 80° C. then (CH₃COO)₃Gd (1.7 g)was added and the solution kept at 80° C. for 3 h. The reaction mixturewas evaporated and the residue was purified by flash chromatography(CH₂Cl₂/MeOH/H₂O=5/5/1 (v/v/v)) to give the title compound (1.53 g; 1.8mmol). Yield 12%.HPLC: 100% (area %); Weight loss (120° C.): 4.96%. TheMS and IR spectra were consistent with the postulated structure.Elemental analysis (%): C H N Gd Calcd. 49.98 7.55 6.66 18.69 Found50.24 7.63 6.61 18.39

EXAMPLE 7[10-[2-Hydroxy-3-[[[2-(Octadecyloxy)-1-[(octadecyloxy)methyl]-ethoxy]-acetyl]oxy]propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0153]

[0154] A) 1,3-Bis(octadecyloxy)-2-propanol (C.A.S. Registry No.18794-74-6)

[0155] Epichlorohydrin (4.6 g; 50 mmol) was added in 5 min to excessstearyl alcohol (117 g; 433 mmol) at 70° C. in the presence of 80% NaHmineral oil dispersion (1.65 g; 55 mmol) (commercial product). Themixture was stirred for 6 h, then cooled to room temperature and treatedwith Et₂O (2 L). The mixture was filtered and the solution evaporated.The crude product was crystallized four times from acetone to give thedesired compound(16.5 g; 27.6 mmol). Yield 55%. K.F.: −0.1%. The ¹H-NMR,¹³C-NMR, MS and IR spectra were consistent with the above structure.Elemental analysis (%): C H Calcd. 78.46 13.51 Found 78.63 13.61

[0156] B) (2-(Octadecyloxy)-1-[(octadecyloxy)methyl]ethoxy)-acetic acid(C.A.S. Registry No. 79979-56-9)

[0157] 80% NaH mineral oil dispersion (2.65 g; 88 mmol) was added undernitrogen atmosphere to a solution of 1,3-bis(octadecyloxy)-2-propanol(6.92 g; 11.6 mmol) in THF (200 mL). The mixture was heated to refluxand a solution of BrCH₂COOH (8.1 g; 58 mmol) in THF (50 mL) was addeddropwise in 30 min. After another 30 min, MeOH was added, then thesolvent was evaporated. The residue was dissolved in Et₂O, washed with0.1 N HCl, dried and evaporated. The crude product was crystallizedtwice from EtOAc to give the desired compound (5.7 g; 8.7 mmol). Yield75%. K.F.: 0.47%; the ¹H-NMR, ¹³C-NMR, MS and IR spectra were consistentwith the foregoing structure. Elemental analysis (%): C H Calcd. 75.1712.62 Found 75.13 13.01 anhydrous

[0158] C) The title compound was then prepared, starting from[2-(octadecyloxy)-1-[(octadecyloxy)methyl]ethoxy]acetic acid and theintermediate (A) of Example 6, according to the synthetic methodreported in Example 6.

EXAMPLE 8[6,9-Bis(carboxymethyl)-3-[2-(octadecylamino)-2-oxoet-hyl]-11-oxo-3,6,9,12-tetraazatriacontanoato(3-)]gadolinium

[0159]

[0160] A)6,9-Bis(carboxymethyl)-3-[2-(octadecylamino)-2-oxoethyl]-11-oxo-3,6,9,12-tetraazatriacontanoicacid (C.A.S. Registry Number 135546-68-8)

[0161] The product was synthesized following the procedure F. Jasanadaand F. Nepveu in Tetrahedron Lett. 33 (1992), 5745-5748. KF: 0.54%. The¹H-NNR, ¹³C-NMR, MS and IR spectra were consistent with the foregoingstructure. Elemental analysis (%): C H N Calcd. 67.0 10.91 7.81 Found67.1 11.31 7.78 anhydrous

[0162] B)[6,9-Bis(carboxymethyl)-3-[2-(octadecylamino)-2-oxoethyl]-11-oxo-3,6,9,12-tetraazatriacontanoato(3-)]qadolinium

[0163] This product was synthetized according to G. W. Kabalka et al.Magn. Reson. Med. 19 (1991), 406-415. Yield 82%. Weight loss (130° C.):5.95%. The MS and IR spectra were consistent with the desired structure.Elemental analysis (%): C H N Gd Calcd. 57.16 9.02 6.67 14.97 Found57.09 9.37 6.57 14.85

EXAMPLE 96,9-Bis(carboxymethyl)-3-(2-oxo-6,9,12,15,18,21,24-hep-taoxa-3-azapentacosyl)-11-oxo-15,18,21,24,27,30,33-heptaoxa-3,6,9,12-tetraazatetratriacontanoato(3-)]gadolinium

[0164]

[0165] A)6,9-Bis(carboxymethyl)-3-(2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azapentacosyl)-11-oxo-15,18,21,24,27,30,33-heptaoxa-3,6,9,12-tetraazatetratriacontanoic acid

[0166] N,N-Bis[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine (commercialproduct) (8.93 g; 25 mmol) was added to a solution of2,5,8,11,14,17,20-heptaoxadocosan-22-amine (prepared according to WO95/17380) (16.97 g; 50 mmol) in DMF (250 mL) at room temperature. After2 h, the reaction mixture was evaporated and the residue was dissolvedin H₂O and 6N HCl. The solution (pH 2) was loaded onto an Amberlite⁵⁸XAD-1600 resin column and eluted with a CH₃CN/H₂O gradient. The productstarts eluting with 10% CH₃CN.

[0167] The fractions containing the product were evaporated and driedunder reduced pressure to give the desired product (14 g; 13.5 mmol).Yield 54%. HPLC: 96% (area %). K.F.: 1.22%. The ¹H-NMR, ¹³C-NMR, MS andIR spectra were consistent with the above structure. Elemental analysis(%): C H N Calcd. 51.00 8.27 6.76 Found 51.55 8.35 6.81 anhydrous

[0168] B)6,9-Bis(carboxymethyl)-3-(2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azapentacosyl)-11-oxo-15,18,21,24,27,30,33-hepta-oxa-3,6,9,12-tetraazatetratriacontanoato(3-)]gadolinium

[0169] Gd₂O₃ (1.86 g; 5.1 mmol) was added to a solution of the freeligand from the previous preparation (10.62 g; 10.2 mmol) in H₂O (200mL) and the resulting suspension was heated at 50° C. for 7 h. Thereaction mixture was filtered through a Millipore® apparatus (HA 0.45 μmfilter); the filtrate was evaporated and dried under reduced pressure togive the title compound (11.9 g; 10 mmol). Yield 98%/. Free ligand(0.001 M GdCl₃): 0.05% (w/w). HPLC: 98% (area %). K.F.: 1.69%. Weightloss (120° C.): 1.58%. The MS and IR spectra were consistent with thestructure. Elemental analysis (%): C H N Gd Calcd. 44.40 6.94 5.88 13.21Found 44.45 7.13 5.91 13.23 anhydrous

EXAMPLE 10[6,9-Bis(carboxymethyl)-3-(2,16-dioxo-6,9,12-trioxa-3,15-diazaririacontanyl)-11,25-dioxo-15,18,21-trioxa-3,6,9,12,24-pentaazadoetraontanoato(3-)]gadolinium

[0170]

[0171] A) 1-[(1-Oxooctadecyl)oxy]-2,5-pyrrolidinedione (C.A.S. RegistryNumber 14464-32-5)

[0172] This compound was synthetized according to M. Shinitzky and R.Haimovitz in J. Am. Chem. Soc. 115 (1993), 12545-12549, and Y. Lapidot,S. Rappoport and Y. Wolman in J. Lipid Res. 8 (1967), 142-145. Yield:86%. K.F.: <0.1%. The ¹H-NMR, ¹³C-NMR and IR spectra were consistentwith the structure. Elemental analysis (%): C H N Calcd. 69.25 10.303.67 Found 69.46 10.77 3.85

[0173] B) 3,6,9-Trioxaundecane-1,11-diamine (C.A.S. Registry No.929-75-9)

[0174] This compound was prepared according to the method disclosed inLiebigs Ann. Chem. 2 (1990), 129-143. Elemental analysis (%): C N Calcd.49.98 14.57 Found 49.68 14.23

[0175] C) 13-Oxo-3,6,9-trioxa-12-azatriacontanylamineH₃₅C₁₇-CONH-[(CH₂)₂-O]₃-(CH₂)₂-NH₂

[0176] 1-[(1-Oxooctadecyl)oxy]-2,5-pyrrolidinedione (3.87 g; 10.1 mmol)in CHCl₃ (600 mL) was added dropwise in 6 h to a solution of3,6,9-trioxaundecane-1,11-diamine (19.8 g; 103 mmol) in CHCl₃ (100 mL)at 20° C. The reaction mixture was evaporated, the residue treated withCH₂Cl₂ (50 mL) and the suspended solid filtered off. The solution wasevaporated, the residue treated with H₂O and extracted with EtOAc. Theorganic phases were combined and dried. Concentration to small volumeled to the precipitation of the desired compound (4.04 g; 8.8 mmol)which was collected by filtration. Yield 87%. The ¹H-NMR, ¹³C-NMR, MSand IR spectra were consistent with the proposed structure. Elementalanalysis (%): C H N Calcd. 68.08 11.87 6.11 Found 67.94 11.91 5.92anhydrous

[0177] D)6,9-Bis(carboxymethyl)-3-(2,16-dioxo-6,9,12-trioxa-3,15-diazatritriacontanyl)-11,25-dioxo-15,18,21-trioxa-3,6,9,12,24-pentaazadotetracontanoicacid

[0178] N,N-Bis[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine (commercialproduct) (6.45 g; 18 mmol) was added to a solution of12-aza-13-oxo-3,6,9-trioxatriacontanylamine (16.57 g; 36 mmol) in DMF(300 mL) at 70° C. After 2 h the mixture was cooled to room temperatureto give a precipitate that was filtered and washed with acetone. Thecrude was crystallized twice from acetone. The solid was filtered,washed with acetone and dried under reduced pressure to give the desiredcompound (17.35 g; 13.6 mmol). Yield 75%. HPLC: 98% (area %); K.F.:0.99%. Weigth loss (120° C.): 0.95%. The ¹H-NMR, ¹³C-NMR, MS and IRspectra were consistent with the above structure. Elemental analysis(%): C H N Calcd. 62.19 10.04 7.69 Found 62.36 10.00 7.55 anhydrous

[0179] E)[6,9-Bis(carboxymethyl)-3-(2,16-dioxo-6,9,12-trioxa-3,15-diazatritriacontanyl)-11,25-dioxo-15,18,21-trioxa-3,6,9,12,24-pentaazadotetracontanoato(3-)]gadolinium

[0180] (CH₃COO)₃Gd.4H₂O (4.06 g; 10 mmol) was added to a solution of theproduct from the previous preparation (12.75 g; 10 mmol) in MeOH (600mL) at 50° C. After 2 h the clear solution was evaporated and driedunder reduced pressure to give the title compound (12.3 g; 8.6 mmol).Yield 86%. Free ligand (0.001 M GdCl₃): 0.02 (w/w). HPLC: 100% (area %);K.F.: 1.90%. Weight loss (120° C.) 3.02%. The MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Gd Calcd.55.47 8.75 6.86 11.00 Found 55.49 8.88 6.73 10.66 anhydrous

EXAMPLE 11[6,9-Bis(carboxymethyl)-3-(2,14-dioxo-18,21,24,27,30,33,36-heptaoxa-3,15-diazaheptatriacontanyl)-11,23-dioxo-27,30,33,36,39,-42,45-heptaoxa-3,6,9,12,24-pentaazahexatetracontanoato(3-)]gadolinium

[0181]

[0182] A) 11-[[(1,1-Dimethylethoxy)carbonyl]amino]undecanoic acid(C.A.S. Registry No. 10436-25-6)

[0183] 11-Aminoundecanoic acid (3 g; 14.9 mmol) was suspended in a 10%solution of Et₃N in MeOH (200 mL); dicarbonic acidbis(1,1-dimethylethyl)ester (Boc₂O) (3.58 g; 16.4 mmol) was added andthe mixture was heated to 50° C. for 15 min. As soon as the aminoaciddissolved, reaction was complete. After evaporation of the solvent underreduced pressure, the triethylammonium salt of the product was treatedwith a 20% solution of citric acid in H₂O and the free acid wasextracted with EtOAc. The organic phase was separated, dried over Na₂SO₄and then evaporated under reduced pressure to give the desired compound(4.45 g; 14.1 mmol). Yield 95%. HPLC: 97% (area %); K.F.: <0.1%. Weightloss (60° C.): 0.83%. The ¹³C-NMR, MS and IR spectra were consistentwith the proposed structure. Elemental analysis (%): C H N Calcd. 63.7610.37 4.65 Found 63.73 10.38 4.63

[0184] B) 11-Amino-N-(3,6,9,12,15,18,21-heptaoxadocosyl)-undecanamide

[0185] To a stirred mixture of11-[[(1,1-dimethyl-ethoxy)carbonyl]amino]undecanoic acid (8.8 g; 29.2mmol), 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (prepared accor-dingto WO 95/17380) (10.9 g; 32.1 mmol) and diethyl cyanophosphonate (DEPC)(5.2 g; 32.1 mmol; 4.9 mL) in DMF (200 mL), maintained at 0° C., Et₃N(3.3 g; 32.1 mmol; 4.5 mL) was added over 1 h. The reaction mixture wasstirred at 0° C. for 30 min and then at room temperature for 1 h. Thesolvent was evaporated under reduced pressure, the crude was dissolvedin 1.2 N HCl in MeOH and the resulting solution was stirred overnight.

[0186] (In an analogous preparation the above-mentioned crude waspurified by washing the solution of the product in EtOAc with 5% aq.NaHCO₃ and identified as11-[[(1,1-dimethylethoxy)carbonyl]amino]-N-(3,6,9,12,15,18,21-heptaoxadocosyl)undecanamide:HPLC: 91% (area %). Weight loss (120° C.): 0.71%; K.F.: 0.64% Elementalanalysis (%): C H N Calcd. 59.78 10.03 4.50 Found 59.59 10.48 4.51Anhydrous

[0187] The ¹³C-NMR, MS and IR spectra were consistent with thestructure).

[0188] After evaporation of the solvent under reduced pressure, theresidue was dissolved in a saturated solution of NaHCO₃ and then washedwith EtOAc. The aqueous phase was separated and acidified with 1N HCluntil precipitation of the product occurred; the latter was filtered togive 11-amino-N-(3,6,9,12,15,18,21-heptaoxadocosyl)undecanamide salifiedwith 1/3 HCl (11.9 g; 22.3 mmol). Yield 76%. HPLC: 95% (area %). K.F.:0.30%. The ¹³C-NMR, MS and IR spectra were consistent with the foregoingstructure. Elemental analysis (%): C H N Cl Calcd. 58.38 10.24 5.24 2.09Found 58.05 10.13 5.15 2.12 anhydrous

[0189] C)6,9-Bis(carboxymethyl)-3-(2,14-dioxo-18,21,24,27,30,33,36-heptaoxa-3,15-diazaheptatriacontanyl)-11,23-dioxo-27,30,33,36,39,42,45-heptaoxa-3,6,9,12,24-pentaazahexatetracontanoicacid

[0190] N,N-Bis[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine (3.8 g; 10.6mmol) (commercial product) was added to a suspension of11-amino-N-(3,6,9,12,15,18,21-heptaoxadocosyl) undecan-amide (11.1 g;21.1 mmol) in DMF (200 mL). After 15 min the reaction mixture becameclear and the conversion was complete. The solvent was evaporated underreduced pressure, the residue was dissolved in CHCl₃ and washed withH₂O. The organic phase was separated, dried over Na₂ SO₄ and thenevaporated under reduced pressure. The solid was dissolved in H₂O andloaded onto an Amberlite® XAD-7 HP resin column (700 mL) and eluted witha CH₃CN/H₂O gradient. The fraction containing the product was evaporatedto give the desired compound (11 g; 7.8 mmol). Yield 74%. HPLC: 85%(area %); K.F. 0.49%. Weight loss (120° C.): 0.45%. The ¹³C-NMR, MS andIR spectra were consistent with the structure. Elemental analysis (%): CH N Calcd. 56.51 9.13 6.99 Found 56.36 9.12 7.02

[0191] D)[6,9-Bis(carboxymethyl)-3-(2,14-dioxo-18,21,24,27,30,33,36-heptaoxa-3,15-diazaheptatriacontanyl)-11,23-dioxo-27,30,33,36,39,-42,45-heptaoxa-3,6,9,12,24-pentaazahexatetracontanoato(3-)]gadolinium

[0192] Gd₂O₃ (0.9 g; 2.5 mmol) was added to a solution of the freeligand from the previous preparation (7 g; 5 mmol) in EtOH (100 mL) andH₂O (150 mL), the resulting suspension was heated at 65° C. for 1 h. Thereaction mixture was filtered through a Millipore® apparatus (HVLP type;0.45 μm filter); the filtrate was evaporated under reduced pressure togive the title compound (7.5 g; 4.74 mmol). Yield 95%. Free gadolinium(0.001 M Na₂EDTA): <0.01% (w/w). HPLC: 96% (area %); K.F.: 1.69%. The MSand IR spectra were consistent with the structure. Elemental analysis(%): C H N Gd Calcd. 50.91 8.03 6.30 10.10 Found 51.06 7.98 6.52  9.94anhydrous

EXAMPLE 12[[6,9-Bis(carboxymethyl)-16-hydroxy-3-(7-hydroxy-2,7-dioxo-6,8-dioxa-3-aza-7-phosphatetracosanyl)-11,16-dioxo-15,17-dioxa-3,6,9,12-tetraaza-16-phosphatritriacontanoato(5-)]gadolinate(2-)]disodiumsalt

[0193]

[0194] A) Phosphoric acid mono(2-aminoethyl) monohexadecyl ester [C.A.S.Registry Number 57303-02-3]

[0195] A solution of hexadecyl alcohol (26.2 g; 108 mmol) in THF (100mL) was added dropwise in 30 min to a solution of POCl₃ (16.56 g; 108mmol) and Et₃N (12.35 g; 122 mmol) in THF (200 mL) at 0° C. After 5 mina solution of ethanolamine (7.2 g; 118 mmol) and Et₃N (43.51 g; 430mmol) in THF (60 mL) was added dropwise in 60 min. at 0° C. The reactionmixture was allowed to reach room temperature in 3 h; then it was heatedat 40° C. and HCl 10% (100 mL) was added. After 2 h the mixture wascooled to room temperature and addition of H₂O (200 mL) afforded aprecipitate that was filtered, washed with H₂O and dried under reducedpressure to give the desired product (33.4 g; 91 mmol). Yield 85%. K.F.:0.53%. The ¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with thestructure. Elemental analysis (%): C H N P Calcd. 59.15 11.03 3.83 8.47Found 59.07 11.41 3.76 8.02 anhydrous

[0196] B)6,9-Bis(carboxymethyl)-16-hydroxy-3-(7-hydroxy-2,7-dioxo-6,8-dioxa-3-aza-7-phosphatetracosanyl)-11,16-dioxo-15,17-dioxa-3,6,9,12-tetraaza-16-phosphatritriacontanoicacid

[0197] N,N-Bis[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine (7.15 g; 20mmol) (commercial product) was added to a suspension of phosphoric acidmono(2-aminoethyl) monohexadecyl ester (14.62 g; 40 mmol) in DMF (700mL) at 75° C. to afford a solution after 15 min. After 5 h the reactionmixture was evaporated and the crude was treated with H₂O and 2N HCl togive a solid that was filtered, washed with H₂O and acetone. The solidwas purified by flash chromatography (CH₂Cl₂/MeOH/NH₄OH 25% (w/w)=6/3/1(v/v/v)).

[0198] The fractions containing the product were combined andconcentrated to 200 mL. Acidification with 2N HCl down to pH 1 led tothe formation of a precipitate that was filtered, washed with H₂O andacetone and dried under reduced pressure to give the desired compound(17 g; 15.6 mmol). Yield 78%. Acidic titer (0.1 N NaOH): 95%. HPLC: 99%(area %). Weight loss (120° C.): 3.45%; K.F.: 3.06%. The ¹H-NMR,¹³C-NMR, MS and IR spectra were consistent with the structure. Elementalanalysis (%): C H N P Calcd. 55.18 9.17 6.43 5.69 Found 55.03 8.97 6.255.44 anhydrous

[0199] C)[[6,9-Bis(carboxymethyl)-16-hydroxy-3-(7-hydroxy-2,7-dioxo-6,8-dioxa-3-aza-7-phosphatetracosanyl)-11,16-dioxo-15,17-dioxa-3,6,9,12-tetraaza-16-phosphatritriacontanoato(5-)]gadolinate(2-)]disodiumsalt

[0200] (CH₃COO)₃Gd.4H₂O (4.06 g; 10 mmol) was added to a solution of thefree ligand from the previous preparation (10.88 g; 10 mmol) in MeOH(500 mL) and 1 N NaOH (20 mL; 20 mmol). After 24 h the clear solutionwas evaporated, the residue dissolved in H₂O (200 mL) and the solutionnanofiltered for 16 h.

[0201] The retentate was evaporated and dried under reduced pressure togive the title compound (11 g; 8.6 mmol). Yield 86%. HPLC: 99% (area %).Weight loss (120° C.): 4.33% K.F.: 4.23%. The MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Gd Na PCalcd. 46.68 7.36 5.44 12.22 3.57 4.82 Found 46.50 7.66 5.44 11.99 3.484.47 anhydrous

EXAMPLE 13[6,9-Bis(carboxymethyl)-11,19-dioxo-3-[2-[[2-[2-[(1-oxooctadecyl)oxylethoxy]ethyl]amino]-2-oxoethyl]-15,18-dioxa-3,6,9,12-tetraazahexatriacontanoato(3-)]gadolinium

[0202]

[0203] A) Octadecanoic acid 2-(2-aminoethoxy)ethyl ester hydrochloride

[0204] 1.2 M HCl in CH₃OH (30 mL) was added to a solution of2-(2-aminoethoxy)ethanol (2.1 g; 20 mmol) (commercial product) in CH₃OH(30 mL); after 30 min the solution was evaporated and dried underreduced pressure to give 2-(2-aminoethoxy)ethanol hydrochloride (2.9 g;20 mmol).

[0205] Stearoyl chloride (6.4 g; 21 mmol) (commercial product) was addeddropwise in 5 min to a solution of 2-(2-aminoethoxy)ethanolhydrochloride (2.9 g; 20 mmol) in DMF (50 mL) at room temperature toafford a suspension. After 16 h, the suspension was diluted with acetoneand the precipitated solid filtered and washed with acetone. The crudewas crystallized from EtOAc; the solid was filtered, washed with EtOAcand dried under reduced pressure to give the desired compound (3.3 g; 8mmol). Yield 40%. Acidic titer (0.1 N NaCH): 93%. K.F.: 1.06%. The¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.Elemental analysis (%): C H N Cl Calcd. 64.75 11.36 3.43 8.69 Found64.82 11.41 3.52 8.90 anhydrous

[0206] B)6,9-Bis(carboxymethyl)-11,19-dioxo-3-[2-[[2-[2-[(1-oxooctadecyl)oxy]ethoxy]ethyl]amino]-2-oxoethyl]-15,18-dioxa-3,6,9,12-tetraaza-hexatriacontanoicacid

[0207] N,N-Bis[2-(2,6-dioxo-4-morpholinyl)ethyl]glycine (1.9 g; 5.5mmol) (commercial product) was added to a solution of octadecanoic acid2-(2-aminoethoxy)ethyl ester hydrochloride (4.5 g; 11 mmol) and Et₃N(1.5 g; 14 mmol) in DMF (150 mL) at 60° C. After 5 min the temperaturewas decreased to 45° C. After 2.5 h the mixture was cooled to roomtemperature to give a precipitate that was filtered, washed withacetone/H₂O 15/5, then with acetone. The crude was dissolved in acetoneand 0.1 N HCl and the solution heated at 50° C. After 10 min thesolution was cooled to room temperature to afford a precipitate whichwas filtered, washed with acetone/H₂O 15/5 then with acetone and driedunder reduced pressure to give the desired compound (1.7 g; 1.5 mmol).Yield 27%. HPLC: 100% (area %). HPCE: 100% (area). K.F.: 3.11%. Weightloss (120° C.): 4.49%. The ¹H-NMR, ¹³C-NMR, MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Calcd.63.30 9.98 6.36 Found 63.29 9.95 6.50

[0208] C)[6,9-Bis(carboxymethyl)-11,19-dioxo-3-[2-[[2-[2-[(1-oxooctadecyl)oxy]ethoxy]ethyl]amino]-2-oxoethyl]-15,18-dioxa-3,6,9,12-tetraazahexatriacontanoato(3-)]gadolinium

[0209] (CH₃COO)₃Gd.4H₂O (1.11 g; 2.7 mmol) was added to a solution ofthe free ligand from the previous preparation (3 g; 2.7 mmol) in MeOH(150 mL) at 40° C. After 1 h the clear solution was evaporated and driedunder reduced pressure. The crude was purified by flash chromatography(CH₃OH) to give the title compound (3.2 g; 2.5 mmol). Yield 94%. HPCE:100% (area %). K.F.: 4.07%. Weight loss (120° C.): 4.08%. the MS and IRspectra were consistent with the structure. Elemental analysis (%): C HN Gd Calcd. 55.52 8.52 5.58 12.53 Found 55.50 8.52 5.58 12.62 anhydrous

EXAMPLE 14[[N,N′-[[[2-(Octadecylamino)-2-oxoethyl]imino]di-2,1-ethanediyl]bis[N-(carboxymethyl)glycinato(4-)]]gadolin-ate(1-)]sodiumsalt

[0210]

[0211] A) N,N-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]-ethyl]glycine

[0212] The compound was prepared according to the method disclosed inpatent application WO-A-95/32741.

[0213] B)N,N′-[[[2-(Octadecylamino)-2-oxoethyl]imino]di-2,1-ethanediyl]bis[N-[2-(1,1-dimethylethoxy)-2-oxoethyl]glycine1,1-dimethylethyl ester]

[0214] Isobutyl chloroformate (205 mg; 1.65 mmol; 215 μL) was addeddropwise to a solution of the product from the previous preparation (950mg; 1.54 mmol) and Et₃N (165 mg; 1.65 mmol; 230 μL) in THF (50 mL) at−5° C. and under nitrogen. After 15 min a suspension of octadecylamine(450 mg; 1.65 mmol) in THF (50 mL) was added to the reaction mixture at−5° C. After 20 min the reaction mixture was allowed to rise to roomtemperature and stirred overnight. The suspension was filtered to removethe residual octadecylamine and the solution was evaporated underreduced pressure. The residue was dissolved in Et₂O and the solutionwashed with 5% aq. NaHCO₃. The organic phase was dried over Na₂SO₄ andthen evaporated under reduced pressure. The crude product was purifiedby flash chromatography (CH₂Cl₂/MeOH=100/2 (v/v)) to give the desiredcompound (1.1 g; 1.26 mmol). Yield 80%. HPLC: 94% (area %). The ¹³C-NMR,MS and IR spectra were consistent with the structure.

[0215] C)N,N′-[[[2-(Octadecylamino)-2-oxoethyl]imino]di-2,1-ethanediyl]bis[N-(carboxymethyl)glycine]

[0216] 0.5 M H₂SO₄ (100 mL; 50 mmol) was added dropwise to a solution ofthe tetraester from the previous preparation (21 g; 24.3 mmol) indioxane (150 mL) and the resulting mixture was heated at 90° C. for 4 h.The pH of the solution was adjusted to 5 with 2 N NaOH (10 mL) and thesolvent was evaporated under reduced pressure. The residue was dissolvedin CHCl₃/MeOH (4:1) and the resulting suspension was filtered andevaporated under reduced pressure. The crude product was purified byflash chromatography (CH₂Cl₂/MeOH/NH₄OH 25% (w/w)=6/3/1 (v/v/v)). Theproduct was dissolved in H₂O (150 mL) and 12 N HCl (7 mL) and desaltedby elution through an Amberlite^(□) XAD 7-HP resin column with aCH₃CN/H₂O gradient. The fractions containing the product were evaporatedto give the desired compound (4 g; 6.2 mmol). Yield 25%. HPLC 99% (area%). K.F.: 3.77%. The ¹³C-NMR, MS and IR spectra were consistent with thestructure. Elemental analysis (%): C H N Calcd. 59.60 9.38 8.69 Found58.70 9.21 8.51 anhydrous

[0217] D)[[N,N′-[[[2-(Octadecylamino)-2-oxoethyl]imino]di-2,1-ethanediyl]bis[N-(carboxymethyl)glycinato(4-)]]gadoli-nate(1-)]sodiumsalt

[0218] 1 N NaOH (4.5 mL) was added to a suspension of the free ligandfrom the previous preparation (2.9 g; 4.5 mmol) in 1:1 H₂O/CH₃CN (600mL). A solution of GdCl₃.6H₂O (1.66 g; 4.48 mmol) in H₂O (50 mL) wasadded dropwise to the reaction mixture mantained at pH 6.8 by theaddition of 1 N NaOH (13.5 mL). After 2 h the solution was evaporated;the residue was dissolved in H₂O and desalted by elution through anAmberlite® XAD 7-HP resin column (250 mL) with a H₂O/CH₃CN gradient.

[0219] The fractions containing the product were evaporated. The solidresidue was dissolved in H₂O and the solution eluted through a Dowex®CCR 3LB weak cation exchange resin column (Na⁺ form, 20 mL). The eluatewas evaporated and dried under reduced pressure to give the titlecompound (1.35 g; 1.64 mmol). Yield 35%. HPLC: 100% (area %). K.F.:8.85%. Weight loss (120 ° C.): 8.15%. The MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Gd NaCalcd. 46.81 6.87 6.82 19.15 2.80 Found 46.79 7.03 6.74 18.96 2.68

EXAMPLE 15[N²,N²-Bis[2-[bis(carboxymethyl)amino]ethyl]-N⁶-(1-oxooctadecyl)-L-lysinato(5-)]gadolinate(2-)]disodiumsalt

[0220]

[0221] A)N²,N²-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]-amino]-ethyl]-L-lysine1,1-dimethylethyl ester

[0222] This product was prepared according to Example 2 of WO 98/05626.The ¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with thedisclosed structure. Elemental analysis (%): C H N Calcd. 61.26 9.747.52 Found 61.43 10.25 7.48

[0223] B)N²,N²-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]-ethyl]-N⁶-(1-oxooctadecyl)-L-lysine-(1,1-dimethylethyl)-ester

[0224] Stearoyl chloride (5.45 g; 18 mmol) dissolved in CHCl₃ (60 mL)was added dropwise in 1 h to a solution ofN²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine1,1-dimethylethyl ester (13.5 g; 18 mmol) in CHCl₃ (300 mL) at 0° C.After 10 min the reaction mixture was allowed to rise to roomtemperature and TLC analysis showed the complete conversion of thestarting materials. The solution was washed with 5% aq. NaHCO₃, theorganic phase was separated, dried over Na₂SO₄ and then evaporated underreduced pressure. The crude product was purified by flash chromatography(n-hexane/EtOAc=6/4 (v/v)) to give the desired product (11.3 g; 11.2mmol). Yield 62%. HPLC: 95% (area %). the ¹³C-NMR, MS and IR spectrawere consistent with the structure.

[0225] C)N²,N²-Bis[2-(bis(carboxymethyl)amino]ethyl]-N⁶-(1-oxooctadecyl)-L-lysine

[0226] N², N²-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]-amino]ethyl]-N⁶-(1-oxooctadecyl)-L-lysine1,1-dimethylethyl ester (9 g, 8.9 mmol) was dissolved in 6 N HCl (200mL) and the solution was stirred for 3 days. The reaction mixture wasdirectly loaded onto an Amberlite^(□) XAD 7-HP resin column and desaltedby elution with a CH₃CN/H₂O gradient.

[0227] The solid obtained from the column was completely converted intothe expected acid by treatment with neat CF₃COOH for 2 h. CF₃COOH waseliminated by repeated dilution with CH₂Cl₂ and Et₂O followed each timeby evaporation under reduced pressure. The residue was dried to give thedesired product (3.1 g; 4.06 mmol). Yield 45%. HPLC: 94.4% (area %).Weight loss (120° C.): 4.22%. The ¹³C-NMR, MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Calcd.59.16 9.10 7.67 Found 59.02 9.38 7.63

[0228] D)[N²,N²-Bis[2-[bis(carboxymethyl)amino]ethyl]-N⁶-(1-oxooctadecyl)-L-lysinato(5-)]gadolinate(2-)]disodiumsalt

[0229] The free ligand from the previous preparation (2.16 g; 2.96 mmol)was suspended in H₂O (50 mL) and dissolved by addition of 1 N NaOH (5.9mL). Dropwise addition of a 1 M aq. solution of (CH₃COO)₃Gd (2.96 mL)led to the precipitation of a solid, which was dissolved by addition ofEtOH (150 mL). The solvent was evaporated under reduced pressure to givethe title compound (2.57 g; 2.66 mmol). Yield 90%. HPLC: 99% (area %).K.F.: 3.81%. Weight loss (120° C.) 3.84%. The MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Gd NaCalcd. 46.54 6.62 6.03 16.92 4.95 Found 46.17 6.38 5.67 16.93 5.42

EXAMPLE 16[[N²,N²-Bis[2-[bis(carboxymethyl)amino]ethyl]-N⁶-[[[(3β-cholest-5-en-3-yl]oxy]carbonyl]-L-lysinato(5-)]gadolinate(2-)]disodiumsalt

[0230]

[0231] A)N²,N²-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]-amino]-ethyl]-N-[([(3,8)-cholest-5-en-3-yl]oxy]carbonyl]-L-lysine 1,1-dimethylethylester

[0232] A solution of cholesteryl chloroformate (11 g; 22 mmol)(commercial product) in CHCl₃ (60 mL) was added in 1 h to a solution ofN²,N²-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine1,1-dimethyl ethyl ester (14.9 g; 20 mmol) (prepared according toExample 2 of WO 98/05626) in CHCl₃ (150 mL). The reaction mixture wasstirred overnight. The solution was then washed with 5% aq. NaHCO₃, theorganic phase was separated, dried over Na₂SO₄ and then evaporated underreduced pressure The crude product was purified by flash chromatography(n-Hexane/EtOAc=85/15 (v/v)) to give the desired compound (18.2 g; 15.7mmol). Yield 78%. HPLC: 94% (area %). Weight loss (100° C.): 1.23%. The¹³C-NMR, MS and IR spectra were consistent with the structure. Elementalanalysis (%): C H N Calcd. 68.48 10.10 4.84 Found 68.89 10.08 4.75

[0233] B)N²,N²-Bis[2-[bis(carboxymethyl)amino]ethyl]-N⁶-[[[(3β)-cholest-5-en-3-yl]oxy]carbonyl]-L-lysine

[0234] 12.6 g of the product from the previous preparation (10.88 mmol)were dissolved in formic acid (250 mL) and refluxed for 1.5 h. Thesolution was evaporated under reduced pressure, the crude was suspendedin water, stirred for 30 min and filtered to give the desired compound(7.8 g; 8.5 mmol). Yield 79%. HPLC: >91% (area %). K.F.: 3.73%. The¹³C-NMR, MS and IR spectra were consistent with this structure Elementalanalysis (%): C H N Calcd. 62.99 8.73 6.39 Found 62.70 8.89 6.31

[0235] C)[[N²,N²-Bis[2-[bis(carboxymethyl)amino]ethyl]-N⁶-[[[(3β)-cholest-5-en-3-yl]oxy]carbonyl]-L-lysinato(5-)]gadolinate(2-)]disodiumsalt

[0236] 5.3 g of the free ligand from the previous preparation (5.8 mmol)were suspended in H₂O (300 mL) and dissolved by the addition of 2 N NaOH(5.8 mL). A solution of GdCl₃.6H₂O (2.16 g; 5.8 mmol) in H₂O (10 mL) wasadded dropwise to the reaction mixture mantained at pH 6.8 by theaddition of 2 N NaOH (8.7 mL). After 1 h the solution was concentratedto 50 mL, the addition of CH₃CN led to the precipitation of the titlecompound, which was filtered and dried (4.94 g; 4.60 mmol). Yield 79%.HPLC: >89% (area %). K.F.: 10.12%. Weight loss (120° C.): 11.89%. The MSand IR spectra were consistent with the structure. Elemental analysis(%): C H N Gd Na Calcd 51.38 6.66 5.21 14.62 4.28 Found 51.47 6.83 5.1714.51 4.02

EXAMPLE 17[10-[[2-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0237]

[0238] A) N-Octadecyl-1-octadecanamine (dioctadecylamine) (C.A.S.Registry No. 112-99-2.

[0239] A1) Dioctadecylcyanamide (C.A.S. Registry No. 113576-09-3)

[0240] Cyanamide (5 g; 119 mmol) was added to stirred 50% aq. NaOH (100g). The mixture was cooled to 25° C., then a solution of Aliquat^(□) 336(trioctylmethylammonium chloride) (2.42 g; 6 mmol) (commercial product)and 1-bromooctadecane (40.37 g; 121 mmol) in toluene (50 mL) was added.The mixture was vigorously stirred at 55° C. for 6 h. The organic phasewas separated and evaporated to give the desired compound (38 g). Thecrude product was used in the hydrolysis step without any furtherpurification. K.F.: <0.1%. The 1H-NMR, ¹³C-NMR, MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Calcd.81.24 13.64 5.12 Found 80.86 13.91 5.19

[0241] A2) N-Octadecyl-1-octadecanamine (dioctadecylamine)

[0242] The crude dioctadecylcyanamide (38 g) was suspended in 2.75 MH₂SO₄ (150 mL) and the mixture was refluxed for 2.5 h. After cooling toroom temperature H₂O (100 mL), 30% NaOH (100 mL) and CHCl₃ (300 mL) wereadded. The organic phase was separated, dried and evaporated. The solidresidue was suspended in Et₂O and stirred for 1 h. The solid wasfiltered and washed with to give the desired compound (15.3 g; 29.3mmol). Yield 48%. K.F.: 0.20%. The ¹H-NMR, ¹³C-NMR, MS and IR spectrawere consistent with the structure. Elemental analysis (%): C H N Calcd.82.83 14.48 2.68 Found 82.61 14.65 2.69 anhydrous

[0243] B) 2-Hydroxy-N,N-dioctadecylacetamide

[0244] (Acetyloxy)acetyl chloride (3.8 g; 28.1 mmol) (commercialproduct) dissolved in CHCl₃ (150 mL) was added dropwise to a solution ofdioctadecylamine (13.3 g; 25.5 mmol) and Et₃N (3.9 mL; 28.1 mmol) inCHCl₃ (350 mL) and the solution was stirred at room temperatureovernight. MeOH (250 mL) and 2 N NaOH (50 mL) were added to thesolution. H₂O was added to the reaction mixture and a two phase systemwas obtained. The lower organic layer was separated and evaporated. Thesolid residue was suspended in n-hexane and filtered to give the desiredcompound (12.1 g;20.9 mmol). Yield 82%. HPLC: 96% (area %). K.F.: <0.1%.The ¹³C-NMR, MS and IR spectra were consistent with the structure.Elemental analysis (%): C H N Calcd. 78.69 13.38 2.41 Found 78.56 13.442.44

[0245] C) N-[(Phenylmethoxy)carbonyl]glycine[2-(dioctadecylamino)-2-oxoethyl]ester

[0246] A solution of DCC (2.1 g; 10.3 mmol) in CHCl₃ (50 mL) was addeddropwise to a solution of 2-hydroxy-N,N-dioctadecylacetamide (5 g; 8.6mmol) and Z-glycine (2 g; 9.5 mmol) in CHCl₃ (250 mL). DMAP (0.1 g; 0.9mmol) was added to the resulting solution. After 1 h the reactionmixture was filtered and the solvent was evaporated. The crude waspurified by flash chromatography (n-Hexane/EtOAc=7/3 (v/v)) to give thedesired compound (5.6 g; 7.3 mmol). Yield 84%. HPLC: 99% (area %). K.F.:<0.1%. The ¹³C-NMR, MS and IR spectra were consistent with thestructure. Elemental analysis (%): C H N Calcd. 74.76 11.24 3.63 Found75.45 11.47 3.68

[0247] D) Glycine [2-(dioctadecylamino)-2-oxoethyl]ester hydrochloride

[0248] 10% Pd/C (150 mg) was added to a solution ofN-[(phenylmethoxy)-carbonyl]glycine[2-(dioctadecylamino)-2-oxoethyl]ester (1.2 g; 1.4 mmol) in EtOAc (100mL) and the suspension was stirred for 3 h under hydrogen atmosphere atroom temperature. After filtration (through a Millipore® filter FT 0.45μm) 1.2 M HCl in MeOH (1.3 mL; 1.6 mmol) was added dropwise to theresulting solution obtaining the precipitation of a white solid that wasfiltered to give the desired compound (830 mg; 1.2 mmol).Yield 86%.HPLC: 100% (area %). K.F.: 0.22%. The MS and IR spectra were consistentwith the structure. Elemental analysis (%): C H N Cl Calcd. 71.33 12.484.16 5.26 Found 71.72 12.48 4.30 5.30 anhydrous

[0249] E) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(phenylmethyl)ester

[0250] E1) 1,4,7,10-Tetraazacyclododecane-1-acetic acid(1,1dimethylethyl)ester

[0251] A solution of t-butyl bromoacetate (25.3 g; 130 mmol) in CHCl₃(500 mL) (commercial product) was added dropwise in 7 h to a solution of1,4,7,10-tetraazacyclododecane (112.3 g; 650 mmol) (commercial product)in CHCl₃ (2 L) maintained under nitrogen at room temperature. After 14 hthe solution was concentrated to 800 mL, washed with H₂O, dried andevaporated to give the desired compound (39 g; 129 mmol). Yield 99%. GC:92% (area %). K.F.: 0.42%. The ¹³C-NMR, MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Na ClCalcd. 56.20 10.06 18.56 = = Found 56.36 10.34 18.84 <0.1 4.56 anhydrous

[0252] E2) 1,4,7,10-Tetraazacyclododecane-1,4,7-tetraacetic acid-(1,1-dimethyl-ethyl)tris(phenylmethyl)ester

[0253] A solution of 1,4,7,10-tetraazacyclododecane-1-acetic acid(1,1-dimethylethyl)ester (36 g; 126 mmol) in DMF (200 mL) was addeddropwise in 7 h to a suspension of benzyl bromoacetate (94.96 g; 414mmol) and K2CO₃ (86.8 g; 628 mmol) in DMF (250 mL) maintained undernitrogen at room temperature. After 14 h the suspension was filtered *iand the solution evaporated to dryness. The residue was dissolved inEtOAc, washed with H₂O, then with brine. The organic phase was i*separated, dried over Na₂SO₄ and evaporated. The residue was purified byflash chromatography (CH₂Cl₂/MeOH=15/1 (v/v)) to give the desiredcompound (51 g; 65 mmol). Yield 51%. HPLC: 90% (area %). K.F.: 0.48%.The ¹³C-NMR, MS and IR spectra were consistent with the structure.Elemental analysis (%): C H N Na Cl Calcd. 62.38 6.91 7.10 2.91 4.49Found 61.77 6.74 6.90 2.90 4.95 anhydrous

[0254] E3) 1,4,7,10-Tetraazacyclododecane-1,4,7-tetraacetic acidtris(phenyl-methyl)ester

[0255] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid(1,1-dimethyl-ethyl)tris(phenylmethyl)ester adduct with NaCl (47.11 g;60 mmol) was dissolved in dioxane (500 mL). The solution was treatedwith 12 N HCl (500 mL) under nitrogen at room temperature, obtaining aprecipitate. After 16 h the suspension was evaporated and the residuedissolved in H₂O by ultrasound sonication. The solution (pH 2) wasloaded onto an Amberlite® XAD-1600 resin column (900 mL) and eluted witha CH₃CN/H₂O gradient. The fractions containing the product wereconcentrated to remove CH₃CN, then extracted with EtOAc. The organicphase was dried over Na₂SO₄ and evaporated. The residue was trituratedwith EtOAc to give the desired compound (21 g; 31 mmol). Yield 52%.HPLC: 99% (area %). K.F.: <0.1%. The ¹³C-NMR, MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Calcd.65.86 6.87 8.30 Found 66.00 7.03 8.33

[0256] F)10-[[2-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]-amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid tris(phenylmethyl)ester

[0257] To a suspension of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(phenylmethyl)ester (2.7 g; 4 mmol) and glycine[2-(dioctadecylamino)-2-oxoethyl]ester hydrochloride (3 g; 4.4 mmol) inCHCl₃ (250 mL) was added DIEA (diisopropylethylamine) (1.5 mL; 8.8mmol). BOP ((benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate, commercial product) (2.2 g; 4.8 mmol) was added tothe resulting solution, which was stirred at room temperature for 2 h.The solvent was evaporated and the solid residue was suspended in 9:1i-PrOH/H₂O and filtered to obtain the desired compound (4.9 g; 3.8mmol). Yield 95%. The ¹³C-NMR, MS and IR spectra were consistent withthe given structure. Elemental analysis (%): C H N Calcd. 71.48 9.666.50 Found 71.23 9.54 6.38

[0258] G)10-[(2-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]-amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

[0259] 10% Pd/C (150 mg) was added to a solution of10-[[2-[2-[2-(dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]amino]-2-oxoethyl]-1,4,7-10-tetraazacyclododecane-1,4,7-triaceticacid tris(phenylmethyl)ester (1.5 g; 1.2 mmol) in CH₃COOH (150 mL) andthe suspension was stirred for 8 h under hydrogen atmosphere at roomtemperature. After filtration (through a Millipore® filter FT 0.45 m)the solvent was evaporated and the residue was dried under reducedpressure to give the desired compound (3 g; 1 mmol). Yield 83%. HPLC:97% (area %) The ¹³C-NMR, MS and IR spectra were consistent with thestructure. Elemental analysis (%): C H N Calcd. 65.72 10.44 8.21 Found65.54 10.22 8.07

[0260] H)[10-[[2-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]-amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0261] The free ligand from the previous preparation (2.2 g; 2.2 mmol)was dissolved in 3:1 EtOH/H₂O (120 mL); a 0.5 M aq. solution of(CH₃COO)₃Gd (4.4 mL) was added dropwise. The resulting solution washeated at 50° C. for 4 h. The solvent was evaporated to give the-titlecompound (2.2 g; 1.9 mmol). Yield 86%. HPLC: 95% (area %). The MS and IRspectra were consistent with the structure. Elemental analysis (%): C HN Gd Calcd. 57.11 8.82 7.14 13.35 Found 56.98 8.74 6.98 13.23

EXAMPLE 18[10-[1-Methylene-14-octadecyl-2,10,13-trioxo-6,9-dioxa-3,14-diazadotriacontanyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0262]

[0263] A) 4-(Dioctadecylamino)-4-oxobutanoic acid (C.A.S. Registry No.37519-63-4)

[0264] A suspension of dioctadecylamine (24.5 g; 47 mmol) (preparedaccording to Example 17, Step A) and succinic anhydride (4.7 g; 47 mmol)in THF (100 mL) was stirred at room temperature for 18 h. Solvent wasremoved by evaporation and the residue was dissolved in CH₂Cl_(2.) Thesolution was washed with 1 N HCl, dried and evaporated. The crude wascrystallized from CH₃CN to give the desired compound (21.8 g; 35 mmol).Yield 75%. K.F.: 0.37%. The ¹³C-NMR, MS and IR spectra were consistentwith the structure. Elemental analysis (%): C H N Calcd. 77.23 12.802.25 Found 77.52 13.38 2.36 anhydrous

[0265] B)[10-[1-Methylene-14-octadecyl-2,10,13-trioxo-6,9-dioxa-3,14-diazadotriacontanyl]-1,4,7,10-tetraazacyclo-dodecane-1,4,7-triacetato(3-)]gadolinium

[0266] 4-(Dioctadecylamino)-4-oxobutanoic acid (7.6 g; 12.3 mmol) inCHCl₃ (100 mL) was added to a solution of10-[2-[[2-(2-hydroxyethoxy)ethyl]amino]-1-(methylene)-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium(8.1 g; 12.3 mmol) (prepared according to Example 2 of WO 96/04259) inDMSO (100 mL), then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimmidehydrochloride (EDCI) (2.6 g; 13.4 mmol) and 4-dimethylaminopyridine(DMAP) (0.75 g; 6.1 mmol) were added and the clear solution was stirredat room temperature. After 24 h more EDCI (2.6 g; 13.4 mmol) was added.After another 24 h CH₃CN and H₂O were added to obtain the precipitationof a solid, which was filtered with a paper filter. The solid wasdissolved in 1/1 CH₂Cl₂/MeOH and the solution loaded onto a silica gelflash column (CH₂Cl₂/MeOH=1/1 (v/v) (5 L); CH₂Cl₂/MeOH/H₂O=5/5/1 (v/v/v)(4 L)). The fractions containing the product were evaporated to give thetitle compound (6 g; 4.7 mmol). Yield 38%. HPLC: 98% (area %). K.F.:3.02%. The MS and IR spectra were consistent with the structure.Elemental analysis (%): C H N Gd Calcd. 58.06 8.87 6.66 12.46 Found58.25 8.92 6.66 12.38 anhydrous

EXAMPLE 19[10-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0267]

[0268] A)10-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid tris-(phenylmethyl)ester

[0269] DBU (820 μL; 5.5 mmol) was added to a suspension of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(phenylmethyl)ester (prepared according to Step E3 of Example 17)(3.71 g; 5.5 mmol) in toluene (350 mL) obtaining a clear solution, then2-bromo-N,N-dioctadecylacetamide (prepared according to Example 5, StepA) (3.9 g; 6.05 mmol) dissolved in toluene (50 mL) was added dropwise.After 2 h the reaction mixture was filtered and the solvent wasevaporated. The crude was suspended in CH₃CN (50 mL) and the insolublewas filtered off with a paper filter. The solution was evaporatedobtaining the desired product (4.63 g; 3.74 mmol). Yield 68%. The¹³C-NMR, MS and IR spectra were consistent with the structure. Elementalanalysis (%): C H N Calcd. 72.83 9.86 5.66 Found 72.67 9.58 5.71anhydrous

[0270] B)10-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

[0271] 10% Pd/C (0.4 g) was added to a solution of the product from theprevious preparation (4.9 g; 4 mmol) in CH₃COOH (400 mL) and thesuspension was stirred for 6 h under hydrogen atmosphere (consumed H₂:270 mL; 12 mmol) at room temperature. After filtration through aMillipore® filter FT 0.45 μm the solvent was evaporated under reducedpressure and the residue was dried (1.3 kPa; NaOH pellets; 35° C.) togive the desired compound (3.1 g; 3.2 mmol). Yield 80%. The ¹³ C-NMR, MSand IR spectra were consistent with the structure. Elemental analysis(%): C H N Calcd. 67.11 10.74 7.25 Found 67.15 10.67 7.11 anhydrous

[0272] C)[10-[2-[2-(Dioctadecylamino)-2-oxoethoxy]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0273] The free ligand from the previous preparation (3.6 g; 3.7 mmol)was dissolved in 2:1 i-PrOH/H₂O (250 mL) and a 0.5 M aq. solution of(CH₃COO)₃Gd (7.4 mL) was added dropwise. The resulting solution washeated at 50° C. for 6 h. The solvent was evaporated to give the titlecompound (3.6 g; 3.2 mmol). Yield 87%.

[0274] MS and IR spectra were consistent with the structure. Elementalanalysis (%): C H N Gd Calcd. 57.88 8.99 6.25 14.03 Found 57.69 8.886.31 13.95 anhydrous

EXAMPLE 20[10-[2-[Bis[2-[(1-oxohexadecyl)oxy]ethyl]amino]-2-oxoethyl]-1,4,7,10tetraazacyclododecane-1,4,7-triacetato(3-)]gadolinium

[0275]

[0276] A) Hexadecanoic acid iminodi-2,1-ethanediyl ester hydrochloride(C.A.S. Registry No. 84454-85-3)

[0277] Palmitoyl chloride (29.8 g; 108.4 mmol) (commercial product) wasadded dropwise in 30 min to a solution of diethanolamine hydrochloride(7 g; 49.4 mmol) (commercial product) in DMF (100 mL). After standingfor 1.5 h a white solid crystallized. MeOH (350 mL) was added and thereaction mixture heated to reflux. After cooling to room temperature therecrystallized product was filtered to give as a white solidhexadecanoic acid iminodi-2,1-ethanediyl ester hydrochloride (15 g; 24.2mmol). Yield 49%. K.F.: 0.4%. The ¹³C-NMR, MS and IR spectra wereconsistent with the structure. Elemental analysis (%): C H N Cl Calcd.69.92 11.73 2.26 5.73 Found 69.79 11.74 2.33 5.82 anhydrous

[0278] B)10-(2-[Bis[2-[(1-oxohexadecyl)oxy]ethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacidtris(phenylmethyl)ester

[0279] A 50% solution of 1-propanephosphonic acid cyclic anhydride (14.4g; 22.6 mmol) in EtOAc (commercial product) was added to a solution of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(phenyl-methyl)ester (15 g; 22.2 mmol) (prepared according to StepE3 of Example 17), hexadecanoic acid iminodi-2,1-ethanediyl esterhydrochloride (14 g; 22.6 mmol) and Et₃N (6.5 mL; 46.6 mmol) in CH₂Cl₂(200 mL). The reaction mixture was stirred at room temperature for 24 h,then more 1-propanephosphonic acid cyclic anhydride (14.4 g; 22.6 mmol)was added. After another 24 h the mixture was washed with brine, driedand evaporated. The residue was purified by flash chromatography(CH₂Cl₂/MeOH=9/1 (v/v)) to give the desired compound (17 g; 13.7 mmol).Yield 62%. HPLC: 96% (area %). The ¹³C-NMR, MS and IR spectra wereconsistent with the proposed structure. Elemental analysis (%): C H NCalcd. 70.78 9.36 5.65 Found 70.57 9.38 5.48 anhydrous

[0280] C)10-[2-[Bis[2-[(1-oxohexadecyl)oxy]ethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

[0281] 10% Pd/C (1.6 g) was added to a solution of10-[2-[bis[2-[(1-oxohexadecyl)oxy]ethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclo-dodecane-1,4,7-triaceticacid tris(phenylmethyl)ester (16 g; 12.9 mmol) in EtOH (500 mL) and thesuspension was stirred for 12 h under hydrogen atmosphere at roomtemperature. After filtration through a Millipore® filter FT 0.45 μm thesolution was evaporated under reduced pressure to give the desiredcompound. Yield 93%. HPLC: 98% (area %). The ¹³C-NMR, MS and IR spectrawere consistent with the structure. Elemental analysis (%): C H N Calcd.64.50 10.10 7.23 Found 64.38 10.01 7.15 anhydrous

[0282] D)[10-[2-[Bis[2-[(1-oxohexadecyl)oxy]ethyl]amino]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetato-(3)]gadolinium

[0283] (CH₃COO)₃Gd.4H₂O (4.63 g; 11.4 mmol) was added to a solution ofthe free ligand from the previous preparation (11 g; 11.4 mmol) in EtOH(500 mL) at 50° C. After 6 h the solution was evaporated and dried underreduced pressure to give the title compound (12.3 g; 11 mmol). Yield96%. HPLC: 99% (area %). The MS and IR spectra were consistent with thestructure. Elemental analysis (%): C H N Gd Calcd. 55.64 8.44 6.24 14.01Found 55.57 8.41 6.12 13.92

1. Compounds either racemic or enantiomeric of the formulae (†) and (IV)and corresponding metal chelates in which n and m

are 1 or 0 but not simultaneously 1, and when n=m=0, R′ is H, and R* isa C₁₂₋₂₅ linear or ramified, saturated or unsaturated, hydrocarbonradical; when n=1 and m=0, R* is H or a C₁₋₃ alkyl or alkylenesubstituent; and R′ is selected from —NHR₃, —NR₄R₅ and —OR₆ where the R₃to R₆ are independently C₁₋₂₅ linear or ramified, saturated orunsaturated, hydrocarbon radicals optionally interrupted by —CO— and/or—O— and optionally terminated by —NR₇R₈ in which R₇ and R₈ areindependently H or C₁₂₋₂₅ hydrocarbon radicals; when n=0 and m=1, R* isH or a C₁₋₃ alkyl or alkylene substituent; and R′ is selected from R₉and CH₂—O—CO—R₉ in which R₉ is a C₁₀₋₃₀ linear or ramified, saturated orunsaturated, hydrocarbon radical optionally interrupted by —NH—, —NR₁₀—,—CO— or —O—, R₁₀ being a lower aliphatic hydrocarbon; and R₁₂ is H or aC₁₂₋₅₀ hydrocarbon radical optionally interrupted by —NH—, —NR₁₀—, —CO—or —O— and optionally terminated by a -cholesteryl residue, and the R₁₃are —OH; or one or two R₁₃ are a —NH— R₁₄ group in which R₁₄ is a C₂₋₃₀linear or ramified, saturated or unsaturated, hydrocarbon radicaloptionally interrupted by —NH—, —NR₁₀—, —CO—, —O—, and/or —OPO(OH)O—,the remaining R₁₃ being —OH.
 2. Compounds encompassed by formula †according to claim 1, having formulae (I), (II) or (III)

wherein R* is defined as in claim 1, R is H or a C₁₋₃ alkyl or alkylenesubstituent; R₁ is selected from —NHR₃, —NR₄R₅ and —OR₆ where the R₃ toR₆ are independently C₁₋₂₅ linear or ramified, saturated or unsaturated,hydrocarbon radicals optionally interrupted by —CO— and/or —O— andoptionally terminated by —NR₇R₈ in which R₇ and R₈ are independently Hor C₁₂₋₂₅ hydrocarbon radicals; R₂ is selected from R₉ and —CH₂—O—CO—R₉in which R₉ is a C₁₀₋₃₀ linear or ramified, saturated or unsaturated,hydrocarbon radical optionally interrupted by one or more —NH—, —NR₁₀—,—CO— or —O—, R₁₀ being a lower aliphatic hydrocarbon.
 3. Compoundsaccording to claim 2 of formula (IIa)

in which R₃ is a C₁₂₋₂₅ linear or ramified, saturated or unsaturated,hydrocarbon radical.
 4. Compounds according to claim 2 of formula (IIb)

in which R₄ and R₅ are independently C₁₂₋₂₅ linear or ramified,saturated or unsaturated, hydrocarbon radicals optionally interrupted by—CO— and/or —O—.
 5. Compounds according to claim 2 of formula (IIc)

in which A is —NH—or —O—, A₁ is a C₁₋₂₀ linear or ramified, saturated orunsaturated, hydrocarbon radicals optionally interrupted by —CO— and/or—O—, and R₇ and R₈ are defined in claim
 1. 6. Compounds according toclaim 2 of formula (IIIa)

in which R is H and R₂ is is a C₁₀₋₃₀ linear or ramified, saturated orunsaturated, hydrocarbon radical optionally interrupted by one or more—NH—, —N—, —CO— or —O—.
 7. Compounds according to claim 2 of formula(IIIB)

in which R is H and R₉ is a C₁₀₋₂₅ linear alkyl, or a C₁₀₋₃₀ linear orramified, saturated or unsaturated, hydrocarbon radical optionallyinterrupted by one or more —N—, —CO— or —O—.
 8. Compounds encompassed byformula (IV) in claim 1, having formula (IVa)

in which the R₁₄ are independently as defined in claim
 1. 9. Compoundsencompassed by formula (IV) in claim 1, having formula (IVb)

in which R₁₄ is a C₁₂₋₂₅ linear or ramified, saturated or unsaturated,hydrocarbon radical.
 10. Compounds according to claim 1 of formula (IV)in which all the R₁₃ are —OH and R₁₂ is defined as in claim
 1. 11.Compounds according to claims 1 to 10, in the form of complex chelateswith paramagnetic metal ions and the salts thereof with physiologicallyacceptable bases selected from primary, secondary, tertiary amines andbasic aminoacids, or inorganic hydroxides of sodium, potassium,magnesium, calcium or mixtures thereof; or with physiologicallyacceptable anions of organic acids selected from acetate, succinate,citrate, fumarate, maleate, oxalate, or inorganic acids selected fromhydrogen halides, sulphates, phosphates, phosphonates and the like; orwith cations or anions of aminoacids selected from lysine, arginine,ornithine, aspartic and glutamic acids, and the like.
 12. NMR imagingcompositions comprising one or more of the compounds of claims 1 to 11,and one or more physiologically acceptable non-ionic surfactants andcarriers.
 13. The compositions of claim 12, wherein the paramagneticmetal ion is selected from Gd(III), Mn(II), Cr(III), Cu(II), Fe(III),Pr(III), Nd(III), Sm(III), Tb(III), Yb(III), Dy(III), Ho(III) andEr(III).
 14. The compositions of claims 12-13, wherein thephysiologically acceptable base is selected from ethanolamine,diethanolamine, morpholine, glucamine, N-methylglucamine,N,N-dimethylglucamine, lysine, arginine, ornithine.
 15. The compositionsof claims 12-14, further comprising one or more amphipatic compounds.16. The compositions of claims 12-14, wherein the non-ionic surfactantis selected from block-copolymers having polyoxyethylene andpolyoxypropylene segments, polyethylene-glycolalkylethers,polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acidesters, and n-alkylglucopyranosides, or n-alkyl maltotriosides.
 17. Thecompositions of claim 16, wherein the surfactant is BRIJ®, Myrj®,Pluronic®, Poloxamer®, Poloxamine®, Synperonic®, Tween®, or mixturesthereof.
 18. The compositions of claims 15-17, wherein the amphipaticcompound is a phosphorus compound, for instance a dialkylglycerophospholipid in which the alkyl group has at least twelve carbonatoms.
 19. The compositions of claim 18, wherein the glycerophospholipidis selected from phosphatidic acid, phosphatidylcholine,phosphatidylethanolamine, phosphatidy-lserine, phosphatidyl-glycerol,phosphatidylinositol, cardiolipin and sphingomyelin.
 20. Thecompositions of claim 18, wherein the phospholipid consists of amono-phosphate ester of a substituted or partially substituted glycerol,at least one functional group of said glycerol being esterified bysaturated or unsaturated aliphatic fatty acid, or etherified bysaturated or unsaturated alcohol, the other two acidic functions of thephosphoric acid being either free or salified with alkali orearth-alkali metals.
 21. The compositions of claim 20, wherein thephospholipid is a monophosphate of a fatty acid glyceride selected fromdimyristoylphosphatidic acid, dipalmitoylphosphatidic acid ordistearoylphosphatidic acid.
 22. The compositions of claims 15, whereinthe amphipatic compound comprises two or more compounds selected fromionic and neutral phospholipids, mono alkyl or alkenyl esters ofphosphoric acid and/or cholesterol, ergosterol, phytosterol, sitosterol,lanosterol and tocopherol.
 23. The compositions of claims 12-22, whereinthe weight ratio of paramagnetic chelate to surfactant in thecomposition is between 1:10 and 10:1, preferably between 1:3 and 3:1.24. The compositions of claims 12-23, wherein the weight ratio ofphospholipids to surfactant in the composition is between 1:10 and 10:1,preferably between 1:2 and 2:1.
 25. A pulverulent formulation comprisingthe ingredients of any of the compositions of claims 12-24 in dry form,said formulation forming, upon dispersion in a physiologicallyacceptable liquid carrier, a dispersion useful as contrast medium forMRI.
 26. An injectable aqueous suspension comprising the compositions ofclaims 12-25 suspended in a physiologically acceptable liquid carrier,useful as NMR imaging contrast medium.
 27. A method for making thecompositions of claims 12-24, characterized by the steps of: a)selecting and suspending a complex of a paramagnetic metal ion with anyone of the chelating agents of formulas (†) to (IVb) claimed in claims1-10, one or more non-ionic surfactants, and optionally one or moreamphipatic compounds in an aqueous phase to form a mixture, and b)energising the mixture by sonicating or microfluidizing to produce anhomogeneous dispersion of the components in micellar form.
 28. Themethod of claim 27, wherein the dispersion of the components in micellarform is sterilized and/or lyophilised.
 29. The method of claims 27-28,wherein the surfactant is added to the mixture after said energising andoptionally repeating the sonication or microfluidization.
 30. Thecompositions of claims 12-24 for use in NMR blood pool imaging of organsin the human or animal body.
 31. Use of the compositions of claims 12-24for the manufacture of an MRI contrast medium.
 32. A two component kitcomprising, as the first component, a dry formulation of claim 25 storedunder an inert atmosphere and, as the second component, aphysiologically acceptable carrier liquid which, when admixed with thefirst component, provides, as a suspension of the two components, aninjectable NMR contrast composition of claims 1-24.
 33. The compositionsof claims 12-24, wherein the MRI responsive components are in micellarform.
 34. The compositions of claim 33, wherein the micelles haveparticle size between 10 and 500 nm, preferably between 50 and 200 nm.